Interleukin-15 receptors

ABSTRACT

There are disclosed Interleukin-15 Receptor (IL-15R) proteins, DNAs and expression vectors encoding IL-15R, and processes for producing IL-15R as products of recombinant cell cultures.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. patent applicationSer. No. 08/435,760, filed May 4, 1995, now pending, which is acontinuation-in-part application of U.S. patent application Ser. No.08/300,903, filed Sep. 6, 1994, now abandoned, which is acontinuation-in-part application of U.S. patent application Ser. No.08/236,919, filed May 6, 1994, now abandoned.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to cytokine receptors,and more specifically, to Interleukin-15 receptors.

[0003] Interleukin-15 (IL-15) is a recently identified cytokine withbiological activities similar to IL-2 (Grabstein et al., Science264:965, 1994). There is approximately 96% nucleotide sequence identityand 96% amino acid sequence identity between human and simian IL-15, andapproximately 81% nucleotide sequence identity and 73% amino acidsequence identity between human and murine IL-15.

[0004] Northern analysis of a variety of human tissues indicated thatIL-15 mRNA is expressed by many human tissues and abundantly by placentaand skeletal muscle. Significant levels of IL-15 mRNA were also observedin other tissues including kidney, lung, liver, and heart. The bestsources of IL-15 mRNA so far observed have been adherent mononuclearcells (monocyte enriched, PBM) and epithelial and fibroblast cell linessuch as CV-1/EBNA and IMTLH. Activated peripheral blood T cells (PBT), arich source of IL-2, express no detectable IL-15 mRNA.

[0005] IL-15 shares many biological properties with Interleukin-2(“IL-2”). These properties include proliferation and activation of humanand murine T cells and the generation of lymphokine activated killercells (LAK), natural killer cells (NK) and cytotoxic T lymphocytes(CTL). IL-15 also can co-stimulate with CD40 ligand (CD40L)proliferation and immunoglobulin secretion by B lymphocytes.

[0006] In view of the shared biological properties with IL-2, tests wereconducted to determine whether IL-15 uses any of the components of theIL-2 receptor. IL-2 cell surface receptors (IL-2R) contain at leastthree subunits, α, β and γ (Toshikazu et al., Science, 257: 379 (1992);see also Minami et al., Annu. Rev. Immunol. 11,245, 1993, for a recentreview). The β and γ chains are required for high affinity IL-2 bindingand IL-2 signaling and are members of the hematopoietin receptorsuperfamily. The α chain (or p55) is a low affinity, non-signalingbinding subunit, and the only cytokine receptor member of a large familyof binding proteins whose members include complement receptor proteins(Perkins et al., Biochemistry 27:4004, 1988; Davie et al., Cold SpringHarb. Symp. Quant. Biol. 51:509, 1986). The γ chain of the IL-2R hasbeen shown recently to be shared by receptors for several othercytokines (IL-4, IL-7, IL-9; (Noguchi et al., Science 262:1877, 1993;Kondo, et al., Science 262:1874, 1993; Kondo et al., Science 263:1453,1994; Russell et al., Science 262:1880, 1993; Russell, et al., Science266:1042, 1994) and designated the common γ chain or γ_(c).

[0007] Several lines of evidence suggest that there is an IL-15 specificbinding protein. For example, an IL-3 dependent murine cell line, 32D(J. S. Greenberger et al., Fed. Proc. 42: 2762 (1983)), expressed thecomplete IL-2R and proliferated in response to IL-2, but cannot bind orrespond to IL-15 (Grabstein et al., supra). Similarly, early murinepre-T cells derived from day 13 fetal liver that lack CD3, CD4 and CD8expression (triple negative, or TN, cells) expressed all three IL-2Rsubunits, proliferated in response to IL-2, but did not bind or respondto IL-15 (Giri et al., EMBO J. 13:2822, 1994). On the other hand,certain human cell types and cell lines (e.g., umbilical veinendothelial cells, fibroblasts and thymic and stromal cells) did notbind IL-2 but bound IL-15 with high affinity (Giri et al., supra).

[0008] Additionally, antibodies directed against the a chain of the IL-2receptor (anti-IL-2Rα) have no effect on IL-15 (Grabstein et al., supra;Giri et al., supra). Antibodies directed against the IL-2Rβ, however,are able to block the activity of IL-15, suggesting that IL-15 uses theB chain of IL-2R. Similarly, some cells require the γ chain of IL-2R forIL-15 signal transduction (Giri et al., supra) IL-15 requires the βchain of the IL-2R for all the biological activities tested, but the αchain of the IL-2R is not required (Giri et al., supra; Grabstein etal., supra). However, prior to the present invention, neither anIL-15-specific binding protein, nor a DNA encoding such protein, hadbeen isolated.

SUMMARY OF THE INVENTION

[0009] The present invention provides isolated Interleukin-15 receptor(IL-15R) and isolated DNA sequences encoding IL-15R, in particular,human and murine IL-15R, or analogs thereof. Preferably, such isolatedDNA sequences are selected from the group consisting of (a) DNAsequences comprising a nucleotide sequence derived from the codingregion of a native IL-15R gene; (b) DNA sequences capable ofhybridization to a DNA of (a) under moderate to high stringencyconditions and that encode biologically active IL-15R; and (c). DNAsequences that are degenerate as a result of the genetic code to a DNAsequence defined in (a) or (b) and that encode biologically activeIL-15R. The present invention also provides recombinant expressionvectors or plasmids and transformed host cells comprising the DNAsequences defined above, recombinant IL-15R proteins produced using therecombinant expression vectors, plasmids or transformed host cells, andprocesses for producing the recombinant IL-15R proteins utilizing theexpression vectors, plasmids or transformed host cells.

[0010] The present invention also provides substantially homogeneouspreparations of IL-15R protein. The present invention also providescompositions for use in assays for IL-15 or IL-15R, purification ofIL-15, or in raising antibodies to IL-15R, comprising effectivequantities of the IL-15R proteins of the present invention.

[0011] These and other aspects of the present invention will becomeevident upon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 illustrates the inhibition of binding of radiolabeled IL-15to CTLL.2 cells by soluble murine IL-15 receptor (HIS-IL15R).

[0013]FIG. 2 presents a sequence alignment between the murine IL-15receptor and the human IL15 receptor (clone W5). The top line representsthe amino acid sequence of human IL-15R; the bottom line represents theamino acid sequence of murine IL-15R. The amino acid sequence has beenseparated into several protein domains:

[0014] 1. signal sequence

[0015] 2. structural domain 1

[0016] 3. proline-rich, flexible hinge region

[0017] 4. structural domain 2

[0018] 5. transmembrane domain

[0019] 6. cytoplasmic domain

[0020] The primary amino acid sequence was also analyzed for predictedstructural characteristics, and found to share common features with agroup of complement factors, and the α subunit of IL-2 receptor. Certainstructural characteristics of the IL-15R are also designated in FIG. 2:

[0021] β: beta sheet

[0022] L: loop

[0023] bold: amino acids conserved among IL-15R and related proteins(i.e., complement control proteins, IL-2 receptor α chain)

[0024] shaded: putative IL-15 binding region

DETAILED DESCRIPTION OF THE INVENTION

[0025] “Interleukin-15 receptor,” “IL-15R” and “IL-15Rα” refer toproteins that are present on many cell types, including cells oflymphoid origin, as well as non-lymphoid cells such as fresh humanendothelial cells, and stromal cells types from bone marrow, fetal liverand thymic epithelium. As used herein, the above terms include analogsor fragments of native and recombinant IL-15R proteins withIL-15-binding activity. Specifically included are truncated, soluble orfusion forms of IL-15R protein as defined below. In the absence of anyspecies designation, IL-15R refers generically to mammalian IL-15R,including but not limited to, human, murine, and bovine IL-15R.Similarly, in the absence of any specific designation for deletionmutants, the term IL-15R means all forms of IL-15R, including mutantsand analogs that possess IL-15R biological activity.

[0026] “Soluble IL-15R” or “sIL-15R” as used in the context of thepresent invention refer to proteins, or substantially equivalentanalogs, that are substantially similar to all or part of theextracellular region of a native IL-15R and are secreted by the hostcell but retain the ability to bind IL-15. Soluble IL-15R proteins mayalso include part of the transmembrane region or part of the cytoplasmicdomain or other sequences, provided that the soluble IL-15R proteins arecapable of being secreted from the host cell in which they are produced.

[0027] The term “isolated” or “purified”, as used in the context of thisspecification to define the purity of IL-15R protein or proteincompositions, means that the protein or protein composition issubstantially free of other proteins of natural or endogenous origin andcontains less than about 1% by mass of protein contaminants residual ofproduction processes. Such compositions, however, can contain otherproteins added as stabilizers, carriers, excipients or co-therapeutics.IL-15R is purified to substantial homogeneity if no other proteins ofnatural or endogenous origin, or protein contaminants residual ofproduction processes, are detected in a polyacrylamide gel by silverstaining.

[0028] The term “substantially similar,” when used to define eitheramino acid or nucleic acid sequences, means that a particular subjectsequence, for example, a mutant sequence, varies from a referencesequence (e.g., a native sequence) by one or more substitutions,deletions, or additions, the net effect of which is to retain biologicalactivity of the IL-15R protein as may be determined, for example, inIL-15R binding assays, such as is described in Example 1 below.Substantially similar analog protein will be greater than about 30percent similar to the corresponding sequence of the native IL-15R. Morepreferably, the analog proteins will be greater than about 80 percentidentical to the corresponding sequence of the native IL-15R. Forfragments of IL-15R proteins, (e.g., soluble IL-15R polypeptides), theterm “80 percent identical” refers to that portion of the referencenative sequence that is found in the IL-15R fragment.

[0029] Computer programs are available for determining the percentidentity between two DNA or amino acid sequences (e.g., between a mutantsequence and a native sequence). One example is the GAP computerprogram, version 6.0, described by Devereux et al., Nucl. Acids Res.12:387 (1984) and available from the University of Wisconsin GeneticsComputer Group (UWGCG). The GAP program uses the alignment method ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), as revised by Smithand Waterman, Adv. Appl. Math 2:482 (1981).

[0030] Alternatively, nucleic acid subunits and analogs are“substantially similar” to the specific native DNA sequences disclosedherein if (a) the DNA sequence is derived from the coding region of anative mammalian IL-15R gene; (b) the DNA sequence is capable ofhybridization to a native IL-15R DNA sequence under moderately stringentconditions (i.e., 50° C., 2×SSC) and encodes biologically active IL-15Rprotein; or (c) the DNA sequence is degenerate as a result of thegenetic code to one of the foregoing native or hybridizing DNA sequencesand encodes a biologically active IL-15R protein. DNA sequences thathybridize to a native IL-15R DNA sequence under conditions of highstringency, and that encode biologically active IL-15R, are alsoencompassed by the present invention. Moderate and high stringencyhybridization conditions are terms understood by the skilled artisan andhave been described in, for example, Sambrook et al., Molecular Cloning:A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring HarborLaboratory Press (1989). IL-15R proteins encoded by the foregoing DNAsequences are provided by the present invention.

[0031] “Recombinant DNA technology” or “recombinant”, as used herein,refers to techniques and processes for producing specific polypeptidesfrom microbial (e.g., bacterial, fungal or yeast) or mammalian cells ororganisms (e.g., transgenics) that have been transformed or transfectedwith cloned or synthetic DNA sequences to enable biosynthesis ofheterologous peptides. Native glycosylation patterns will only beachieved with mammalian cell expression systems. Yeast provide adistinctive glycosylation pattern. Prokaryotic cell expression (e.g., E.coli) will generally produce polypeptides without glycosylation.

[0032] “Biologically active”, as used throughout the specification as acharacteristic of IL-15R, means that a particular molecule sharessufficient amino acid sequence similarity with a native IL-15R proteinto be capable of binding detectable quantities of IL-15, preferably withaffinity similar to native IL-15R.

[0033] A “DNA sequence” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, that hasbeen derived from DNA isolated at least once in substantially pure form(i.e., free of contaminating endogenous materials) and in a quantity orconcentration enabling identification, manipulation, and recovery of itscomponent nucleotide sequences by standard biochemical methods such asthose outlined in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989). Such sequences are preferably provided in the form of an openreading frame uninterrupted by internal nontranslated sequences, orintrons, that are typically present in eukaryotic genes. Sequences ofnon-translated DNA may be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding regions.

[0034] “Nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides. DNA sequences encoding the proteins provided bythis invention may be assembled from cDNA fragments and shortoligonucleotide linkers, or from a series of oligonucleotides, toprovide a synthetic gene that is capable of being expressed in arecombinant transcriptional unit.

[0035] “Recombinant expression vector” refers to a plasmid comprising atranscriptional unit comprising an assembly of (1) a genetic element orelements having a regulatory role in gene expression, for example,promoters or enhancers, (2) a structural or coding sequence that istranscribed into mRNA and translated into protein, and (3) appropriatetranscription and translation initiation and termination sequences.Structural elements intended for use in yeast expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

[0036] “Recombinant rmicrobial expression system” means a substantiallyhomogeneous monoculture of suitable host microorganisms, for example,bacteria such as E. coli or yeast such as S. cerevisiae, that has stablyintegrated a recombinant transcriptional unit into chromosomal DNA orcarries the recombinant transcriptional unit as a component of aresident plasmid. Generally, cells constituting the system are theprogeny of a single ancestral transformant. Recombinant expressionsystems as defined herein will express heterologous protein uponinduction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

[0037] Isolation of DNA Encoding IL-15R

[0038] As shown by Scatchard analysis of iodinated IL-15 binding,activated PBT as well as antigen specific T cell clones express only afew hundred receptors for IL-15. Cells from the murine Th2 CD4⁺ cellclone, D10 (Kaye et al., J. Immunol. 133:1339 (1984)), express up to24,000 IL-15 receptors when cultured with IL-2. A murine DNA sequenceencoding murine IL-15R was isolated from a cDNA library prepared usingstandard methods by reverse transcription of polyadenylated RNA isolatedfrom D10 cells. Transfectants expressing biologically active IL-15R wereinitially identified using a slide autoradiographic technique,substantially as described by Gearing et al., EMBO J. 8:3667 (1989).

[0039] A D10 cDNA library in plasmid pDC304 was prepared as described inLarsen et al., J. Exp. Med., 172:159 (1990). pDC304 is derived frompDC302 previously described by Mosley et al., Cell, 59: 335-348 (1989)by deleting the adenovirus tripartite leader (TPL) in pDC302.

[0040] Using this approach, approximately 20,000 cDNAs were screened inpools of approximately 1000 cDNAs each using the slide autoradiographicmethod until assay of one transfectant pool showed multiple cellsclearly positive for IL-15 binding. This pool was then partitioned intopools of approximately 100 and again screened by slide autoradiographyand a positive pool was identified. Individual colonies from this poolof approximately 100 were screened until a single clone (clone D1-4-D5)was identified that directed synthesis of a surface protein withdetectable L-15 binding activity. This clone was isolated and sequencedto determine the sequence of the murine IL-15R cDNA clone, D1-4-D5. Thecloning vector pDC304 containing the murine IL-15R cDNA clone, D1-4-D5,was deposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852 USA (“ATCC”) in accordance with the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure on Apr. 22, 1994, under accessionnumber ATCC 62659. The deposit was named “D1-4-D5 (pDC304:muIl-15R)” andcomprised an E. coli strain containing a murine IL-15R cDNA insert thatis made up of a 71-bp 5′ noncoding region preceding an open readingframe of 792 bp and a 995-bp 3′ non coding region (the 3′-mostapproximately 200 bp of which is likely to be derived from non-relatedsequence). The nucleotide sequence of the open reading frame isdisclosed in SEQ ID NO: 1. All restrictions on the availability to thepublic of the material deposited will be irrevocably removed upon thegranting of a patent.

[0041] A probe may be constructed from the murine sequence and used toscreen various other mammalian cDNA libraries. cDNA clones thathybridize to the murine probe are then isolated and sequenced.

[0042] Like most mammalian genes, mammalian IL-15R is encoded by amulti-exon gene. IL-15R variants can be attributed to different mRNAsplicing events following transcription or from proteolytic cleavage ofthe IL-15R protein, wherein the IL-15R binding property is retained.Alternative splicing of mRNA may yield a truncated but biologicallyactive IL-15R protein, such as a soluble form of the protein. Variationsattributable to proteolysis include, for example, differences in the N-or C-termini upon expression in different types of host cells, due toproteolytic removal of one or more terminal amino acids from the IL-15Rprotein (generally from 1-5 terminal amino acids). Signal peptides maybe cleaved at different positions in a given protein, resulting invariations of the N-terminal amino acid of the mature protein. TheseIL-15R variants share large regions of identity or similarity with thecDNAs claimed herein and are considered to be within the scope of thepresent invention.

[0043] Proteins and Analogs

[0044] The present invention provides recombinant mammalian IL-15Rpolypeptides. Isolated IL-15R polypeptides of this invention aresubstantially free of other contaminating materials of natural orendogenous origin and contain less than about 1% by mass of proteincontaminants residual of production processes. The IL-15R polypeptidesof this invention are optionally without associated native-patternglycosylation.

[0045] Mammalian IL-15R of the present invention includes, by way ofexample, primate, human, murine, canine, feline, bovine, ovine, equineand porcine IL-15R. The amino acid sequence of a full length murineIL-15R (i.e., including signal peptide, extracellular domain,transmembrane region and cytoplasmic domain) is shown in SEQ ID NOs: 1and 2. The amino acid sequence in SEQ ID NOs: 1 and 2 predicts a type 1membrane protein (i.e., a single transmembrane region with a N-terminalextracellular domain and a C-terminal cytoplasmic domain). The predictedsignal peptide cleavage occurs between amino acids 30 and 31 in SEQ IDNO:2. The predicted transmembrane region includes amino acids 206 to 226in SEQ ID NO:2. Mammalian IL-15R cDNA can be obtained by cross specieshybridization, for example, by using a single stranded probe derivedfrom the murine IL-15R DNA sequence, SEQ ID NO:1, as a hybridizationprobe to isolate IL-15R cDNAs from mammalian cDNA libraries. Theisolated IL-15R cDNAs then can be transfected into expression vectorsand host cells to express the IL-15R proteins.

[0046] Derivatives of IL-15R within the scope of the invention alsoinclude various structural forms of the primary protein that retainbiological activity. Due to the presence of ionizable amino and carboxylgroups, for example, an IL-15R protein may be in the form of acidic orbasic salts, or may be in neutral form. Individual amino acid residuesmay also be modified by oxidation or reduction.

[0047] The primary amino acid structure may be modified by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, lipids, phosphate, acetyl groups and the like, or bycreating amino acid sequence mutants. Covalent derivatives are preparedby linking particular functional groups to IL-15R amino acid side chainsor at the N- or C-termini. Other derivatives of IL-15R within the scopeof this invention include covalent or aggregative conjugates of IL-15Ror its fragments with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.

[0048] When initially expressed in a recombinant system, IL-15R maycomprise a signal or leader polypeptide sequence (native orheterologous) at the N-terminal region of the protein. The signal orleader peptide co-translationally or post-translationally directstransfer of the protein from its site of synthesis to its site offunction outside the cell membrane or wall, and is cleaved from themature protein during the secretion process. Further, using conventionaltechniques, IL-15R polypeptides can be expressed as polypeptide fusionscomprising additional polypeptide sequences, such as Fc or otherimmunoglobulin sequences, linker sequences, or other sequences thatfacilitate purification and identification of IL-15R polypeptides.

[0049] IL-15R derivatives may also be used as immunogens, reagents inreceptor-based immunoassays, or as binding agents for affinitypurification procedures of IL-15 or other binding ligands. IL-15Rderivatives may also be obtained by cross-linking agents, such asm-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, atcysteine and lysine residues. IL-15R proteins may also be covalentlybound through reactive side groups to various insoluble substrates, suchas cyanogen bromide-activated, bisoxirane-activated,carbonyldiimidazole-activated or tosyl-activated agarose structures, orby adsorbing to polyolefin surfaces (with or without glutaraldehydecross-linking). Once bound to a substrate, IL-15R may be used toselectively bind (for purposes of assay or purification) anti-IL-15Rantibodies or IL-15.

[0050] The IL-15R proteins of the present invention encompass proteinshaving amino acid sequences that vary from those of native IL-15Rproteins, but that retain the ability to bind IL-15. Such variantproteins comprise one or more additions, deletions, or substitutions ofamino acids when compared to a native sequence, but exhibit biologicalactivity that is essentially equivalent to that of native IL-15Rprotein. Likewise, the IL-15R-encoding DNA sequences of the presentinvention encompass sequences that comprise one or more additions,deletions, or substitutions of nucleotides when compared to a nativeIL-15R DNA sequence, but that encode an IL-15R protein that isessentially bioequivalent to a native IL-15R protein. Examples of suchvariant amino acid and DNA sequences (the “substantially similar”sequences discussed above) include, but are not limited to, thefollowing.

[0051] Bioequivalent analogs of IL-15R proteins may be constructed by,for example, making various substitutions of residues or sequences ordeleting terminal or internal residues or sequences not needed forbiological activity. For example, cysteine residues not essential forbiological activity can be deleted or replaced with other amino acids toprevent formation of unnecessary or incorrect intramolecular disulfidebridges upon renaturation.

[0052] Another embodiment of the present invention involves modificationof adjacent dibasic amino acid residues to enhance expression of IL-15Rin yeast systems in which KEX2 protease activity is present. Generally,substitutions should be made conservatively; i. e., the most preferredsubstitute amino acids are those having physiochemical characteristicsresembling those of the residue to be replaced. Similarly, when adeletion or insertion strategy is adopted, the potential effect of thedeletion or insertion on biological activity should be considered.

[0053] Substantially similar polypeptide sequences, as defined above,generally comprise a like number of amino acid sequences, althoughC-terminal truncations for the purpose of constructing soluble IL-15Rswill contain fewer amino acid sequences. In order to preserve thebiological activity of IL-15Rs, deletions and substitutions willpreferably result in homologous or conservatively substituted sequences,meaning that a given residue is replaced by a biologically similarresidue. Examples of conservative substitutions include substitution ofone aliphatic residue for another, such as Ile, Val, Leu, or Ala for oneanother, or substitutions of one polar residue for another, such asbetween Lys and Arg; Glu and Asp; or Gln and Asn. Other suchconservative substitutions, for example, substitutions of entire regionshaving similar hydrophobicity characteristics, are well known. Moreover,particular amino acid differences between human, murine and othermammalian IL-15Rs are suggestive of additional conservativesubstitutions that may be made without altering the essential biologicalcharacteristics of IL-15R.

[0054] The present invention includes IL-15R with or without associatednative-pattern glycosylation. IL-15R expressed in yeast or mammalianexpression systems, e.g., COS-7 cells, may be similar or slightlydifferent in molecular weight and glycosylation pattern than the nativemolecules, depending upon the expression system. Expression of IL-15RDNAs in bacteria such as E. coli provides non-glycosylated molecules.Functional mutant analogs of mammalian IL-15R having inactivatedN-glycosylation sites can be produced by oligonucleotide synthesis andligation or by site-specific mutagenesis techniques. These analogproteins can be produced in a homogeneous, reduced-carbohydrate form ingood yield using yeast expression systems. N-glycosylation sites ineukaryotic proteins are characterized by the amino acid tripletAsn-A₁-Z, where A₁ is any amino acid except Pro, and Z is Ser or Thr. Inthis sequence, asparagine provides a side chain amino group for covalentattachment of carbohydrate. Such sites can be eliminated by substitutinganother amino acid for Asn or for residue Z, deleting Asn or Z, orinserting a non-Z amino acid between A₁ and Z, or an amino acid otherthan Asn between Asn and A₁.

[0055] Subunits of IL-15R may be constructed by deleting terminal orinternal residues or sequences. Particularly preferred sequences includethose in which the transmembrane region and intracellular domain ofIL-15R are deleted or substituted with hydrophilic residues tofacilitate secretion of the receptor into the cell culture medium.Soluble IL-15R proteins may also include part of the transmembraneregion, provided that the soluble IL-15R protein is capable of beingsecreted from the cell. The resulting protein is referred to as asoluble IL-15R molecule that retains its ability to bind IL-15. Thepresent invention contemplates such soluble IL-15R constructscorresponding to all or part of the extracellular region of IL-15R. Theresulting soluble IL-15R constructs are then inserted and expressed inappropriate expression vectors and assayed for the ability to bindIL-15, as described in Example 1. Biologically active soluble IL-15Rs(i.e., those which bind IL-15) resulting from such constructions arealso contemplated to be within the scope of the present invention.Soluble IL-15Rs can be used to inhibit IL-15, for example, inameliorating undesired effects of IL-15, in vitro or in vivo. Forexample, significant levels of IL-15 mRNA occur in kidney, lung, liver,and heart, organs that may be transplanted. Soluble IL-15Rs are thuslikely to be useful as IL-15 antagonists in preventing or treating graftrejection. Soluble IL-15Rs can also be used as components ofquantitative or qualitative assays for IL-15, or for affinitypurification of IL-15.

[0056] Mutations in nucleotide sequences constructed for expression ofthe above-described variant or analog IL-15R proteins should, of course,preserve the reading frame phase of the coding sequences and preferablywill not create complementary regions that could hybridize to producesecondary mRNA structures such as loops or hairpins that would adverselyaffect translation of the receptor mRNA. Although a mutation site may bepredetermined, it is not necessary that the nature of the mutation perse be predetermined. For example, in order to select for optimumcharacteristics of mutants at a given site, random mutagenesis may beconducted at the target codon and the expressed IL-15R mutants screenedfor the desired activity.

[0057] Not all mutations in the nucleotide sequence that encodes IL-15Rwill be expressed in the final product. For example, nucleotidesubstitutions may be made to enhance expression, primarily to avoidsecondary structure loops in the transcribed mRNA (see EPA 75,444A,incorporated herein by reference), or to provide codons that are morereadily translated by the selected host, e.g., the well-known E. colipreference codons for E. coli expression (see U.S. Pat. No. 4,425,437,column 6). The known degeneracy of the genetic code permits variation ofa DNA sequence without altering the amino acid sequence, since a givenamino acid may be encoded by more than one codon.

[0058] Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

[0059] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Examples of methods of making the alterations set forth aboveare disclosed by Walder et al., Gene 42:133 (1986); Bauer et al., Gene37:73 (1985); Craik, BioTechniques, 12-19 (1985); Smith et al., GeneticEngineering: Principles and Methods, Plenum Press (1981); and U.S. Pat.Nos. 4,518,584 and 4,737,462.

[0060] The IL-15R proteins of the present invention encompass proteinsencoded by (a) a DNA sequence derived from the coding region of a nativeIL-15R gene or (b) a DNA sequence capable of hybridization to a nativeIL-15R DNA of (a) under moderate to high stringency conditions and thatencodes biologically active IL-15R. IL-15R proteins encoded by a DNAmolecule that varies from the DNA sequences of SEQ ID NO:1, wherein onestrand of the DNA molecule will hybridize to the DNA sequence presentedin SEQ ID NO:1, include, but are not limited to, IL-15R fragments(soluble or membrane-bound) and IL-15R proteins comprising inactivatedN-glycosylation site(s), inactivated KEX2 protease processing site(s),and/or conservative amino acid substitution(s), as described above.IL-15R proteins encoded by DNA derived from other mammalian species,wherein the DNA will hybridize to the murine DNA of SEQ ID NO:1, arealso encompassed.

[0061] Both monovalent forms and polyvalent forms of IL-15R are usefulin the compositions and methods of this invention. Polyvalent formspossess multiple IL-15R binding sites for IL-15 ligand. For example, abivalent soluble IL-15R may consist of two tandem repeats of theextracellular region of IL-15R, separated by a linker region. Two IL-15Rpolypeptides (each capable of binding IL-15) may be joined by anysuitable means, e.g., using one of the commercially availablecross-linking reagents used to attach one polypeptide to another (PierceChemical Co., Rockford, Ill.). Alternatively, a fusion proteincomprising multiple IL-15R polypeptides joined by peptide linkers may beproduced using recombinant DNA technology. Suitable peptide linkerscomprise a chain of amino acids, preferably from 20 to 100 amino acidsin length. The linker advantageously comprises amino acids selected fromthe group consisting of glycine, asparagine, serine, threonine, andalanine. Examples of suitable peptide linkers and the use of suchpeptide linkers are found in U.S. Pat. No. 5,073,627.

[0062] Alternate polyvalent forms may also be constructed, for example,by chemically coupling IL-15R to any clinically acceptable carriermolecule, a polymer selected from the group consisting of Ficoll,polyethylene glycol or dextran using conventional coupling techniques.Alternatively, IL-15R may be chemically coupled to biotin, and thebiotin-IL-15R conjugate then allowed to bind to avidin, resulting intetravalent avidin/biotin/IL-15R molecules. IL-15R may also becovalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) andthe resulting conjugate precipitated with anti-DNP or anti-TNP-IgM, toform decameric conjugates with a valency of 10 for IL-15R binding sites.

[0063] A recombinant chimeric antibody molecule may also be producedhaving IL-15R sequences substituted for the variable domains of eitheror both of the immunoglobulin molecule heavy and light chains and havingunmodified constant region domains. For example, chimeric IL-15R/IgG₁may be produced from two chimeric genes—an IL-15R/human K light chainchimera (IL-15R/CK) and an IL-15R/human γ1 heavy chain chimera(IL-15R/C_(γ-1)). Following transcription and translation of the twochimeric genes, the gene products assemble into a single chimericantibody molecule having IL-15R displayed bivalently. Such polyvalentforms of IL-15R may have enhanced binding affinity for IL-15 ligand.Additional details relating to the construction of such chimericantibody molecules are disclosed in WO 89/09622 and EP 315062.

[0064] Expression of Recombinant IL-15R

[0065] The present invention provides recombinant expression vectors toamplify or express DNA encoding IL-15R. Recombinant expression vectorsare replicable DNA constructs that have synthetic or cDNA-derived DNAfragments encoding mammalian IL-15R or bioequivalent analogs operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, microbial, viral or insect genes. Atranscriptional unit generally comprises an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, transcriptional promoters or enhancers, (2) a structural orcoding sequence that is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements may include an operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sites. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants mayadditionally be incorporated. DNA regions are operably linked when theyare functionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operably linked to DNA for a polypeptideif it is expressed as a precursor that participates in the secretion ofthe polypeptide; a promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; or a ribosome bindingsite is operably linked to a coding sequence if it is positioned so asto permit translation. Generally, operably linked means contiguous and,in the case of secretory leaders, contiguous and in reading frame.Structural elements intended for use in yeast expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

[0066] DNA sequences encoding mammalian IL-15Rs that are to be expressedin a microorganism will preferably contain no introns that couldprematurely terminate transcription of DNA into mRNA. However, prematuretermination of transcription may be desirable, for example, where itwould result in mutants having advantageous C-terminal truncations, forexample, deletion of a transmembrane region to yield a soluble receptornot bound to the cell membrane. Due to code degeneracy, there can beconsiderable variation in nucleotide sequences encoding the same aminoacid sequence. Other embodiments include sequences capable ofhybridizing to SEQ ID NO:1 under at least moderately stringentconditions (50° C., 2×SSC) and other sequences hybridizing or degenerateto those that encode biologically active IL-15R polypeptides.

[0067] Recombinant IL-15R DNA is expressed or amplified in a recombinantexpression system comprising a substantially homogeneous monoculture ofsuitable host microorganisms, for example, bacteria such as E. coli oryeast such as S. cerevisiae, that have stably integrated (bytransformation or transfection) a recombinant transcriptional unit intochromosomal DNA or carry the recombinant transcriptional unit as acomponent of a resident plasmid. Mammalian host cells are preferred forexpressing recombinant IL-15R. Generally, cells constituting the systemare the progeny of a single ancestral transformant. Recombinantexpression systems as defined herein will express heterologous proteinupon induction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

[0068] Transformed host cells are cells that have been transformed ortransfected with IL-15R vectors constructed using recombinant DNAtechniques. Transformed host cells ordinarily express IL-15R, but hostcells transformed for purposes of cloning or amplifying IL-15R DNA donot need to express IL-15R. Expressed IL-15R will be deposited in thecell membrane or secreted into the culture supernatant, depending on theIL-15R DNA selected. Suitable host cells for expression of mammalianIL-15R include prokaryotes, yeast or higher eukaryotic cells under thecontrol of appropriate promoters. Prokaryotes include gram negative orgram positive organisms, for example E. coli or bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems could also be employed toproduce mammalian IL-15R using RNAs derived from the DNA constructs ofthe present invention. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts aredescribed by Pouwels et al., Cloning Vectors: A Laboratory Manual,Elsevier, N.Y. (1985).

[0069] Prokaryotic expression hosts may be used for expression of IL-15Rthat do not require extensive proteolytic and disulfide processing.Prokaryotic expression vectors generally comprise one or more phenotypicselectable markers, for example a gene encoding proteins conferringantibiotic resistance or supplying an autotrophic requirement, and anorigin of replication recognized by the host to ensure amplificationwithin the host. Suitable prokaryotic hosts for transformation includeE. coli, Bacillus subtilis, Salmonella typhimurium, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphyolococcus,although others may also be employed as a matter of choice.

[0070] Useful expression vectors for bacterial use can comprise aselectable marker and bacterial origin of replication derived fromcommercially available plasmids comprising genetic elements of thewell-known cloning vector pBR322 (ATCC 37017). Such commercial vectorsinclude, for example, pKK223-3 and pGEX (Pharmacia Fine Chemicals,Uppsala, Sweden) and pGEMI (Promega Biotec, Madison, Wis., USA). ThesepBR322 “backbone” sections are combined with an appropriate promoter andthe structural sequence to be expressed. E. coli is typicallytransformed using derivatives of pBR322, a plasmid derived from an E.coli species (Bolivar et al., Gene 2:95 (1977)). pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides simplemeans for identifying transformed cells.

[0071] Promoters commonly used in recombinant microbial expressionvectors include the β-lactamase (penicillinase) and lactose promotersystem (Chang et al., Nature 275:615 (1978); and Goeddel et al., Nature281:544 (1979)), the tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057 (1980); and EPA 36,776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412 (1982)). A particularly useful bacterial expressionsystem employs the phage λ P_(L) promoter and cI857ts thermolabilerepressor. Plasmid vectors available from the American Type CultureCollection that incorporate derivatives of the λ P_(L) promoter includeplasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28,resident in E. coli RR1 (ATCC 53082).

[0072] Recombinant IL-15R proteins may also be expressed in yeast hosts,preferably from the Saccharomyces species, such as S. cerevisiae. Yeastof other genera, such as Pichia or Kluyveromyces may also be employed.Yeast vectors will generally contain an origin of replication from the2μ yeast plasmid or an autonomously replicating sequence (ARS),promoter, DNA encoding IL-15R, sequences for polyadenylation andtranscription termination and a selection gene. Preferably, yeastvectors will include an origin of replication and selectable markerpermitting transformation of both yeast and E. coli, e.g., theampicillin resistance gene of E. coli and S. cerevisiae TRP1 or URA3gene, that provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, and a promoter derived from ahighly expressed yeast gene to induce transcription of a structuralsequence downstream. The presence of the TRP1 or URA3 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan oruracil.

[0073] Suitable promoter sequences in yeast vectors include thepromoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman etal., J. Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hesset al., J. Adv. Enzyme Reg. 7:149 (1968); and Holland et al., Biochem.17:4900 (1978)). Suitable vectors and promoters for use in yeastexpression are further described in R. Hitzeman et al., EPA 73,657.

[0074] Preferred yeast vectors can be assembled using DNA sequences frompUC18 for selection and replication in E. coli (Amp^(r) gene and originof replication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al., J. Biol. Chem. 258:2674 (1982) and Beier etal., Nature 300:724 (1982). The yeast α-factor leader, that directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed (see, e.g., Kurjan et al., Cell30:933 (1982); and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330(1984)). The leader sequence may be modified to contain, near its 3′end, one or more useful restriction sites to facilitate fusion of theleader sequence to foreign genes. Suitable yeast transformationprotocols are known to those of skill in the art (see Hinnen et al.,Proc. Natl. Acad. Sci. USA 75:1929 (1978); Sherman et al., LaboratoryCourse Manual for Methods in Yeast Genetics, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1986)).

[0075] Host strains transformed by vectors comprising the ADH2 promotermay be grown for expression in a rich medium consisting of 1% yeastextract, 2% peptone, and 1% or 4% glucose supplemented with 80 μg/mladenine and 80 μg/ml uracil. Derepression of the ADH2 promoter occursupon exhaustion of medium glucose. Crude yeast supernatants areharvested by filtration and held at 4° C. prior to further purification.

[0076] Various mammalian or insect cell culture systems are alsoadvantageously employed to express recombinant protein. Expression ofrecombinant proteins in mammalian cells is particularly preferredbecause such proteins are generally correctly folded, appropriatelymodified and completely functional. Examples of suitable mammalian hostcell lines include the COS-7 lines of monkey kidney cells, described byGluzman, Cell 23:175 (1981), and other cell lines capable of expressinga heterologous gene in an appropriate vector including, for example, Lcells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines.Mammalian expression vectors may comprise nontranscribed elements suchas an origin of replication, a suitable promoter and enhancer linked tothe gene to be expressed, and other 5′ or 3′ flanking nontranscribedsequences, and 5′ or 3′ nontranslated sequences, such as necessaryribosome binding sites, a polyadenylation site, splice donor andacceptor sites, and transcriptional termination sequences. Baculovirussystems for production of heterologous proteins in insect cells arereviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

[0077] The transcriptional and translational control sequences inexpression vectors to be used in transforming vertebrate cells may beprovided by viral sources. For example, commonly used promoters andenhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40(SV40), and human cytomegalovirus. DNA sequences derived from the SV40viral genome, for example, SV40 origin, early and late promoter,enhancer, splice, and polyadenylation sites may be used to provide theother genetic elements required for expression of a heterologous DNAsequence. The early and late promoters are particularly useful becauseboth are obtained easily from the virus as a fragment that also containsthe SV40 viral origin of replication (Fiers et al., Nature 273:113(1978)). Smaller or larger SV40 fragments may also be used, provided theapproximately 250 bp sequence extending from the Hind III site towardthe BglI site located in the viral origin of replication is included.Further, mammalian genomic IL-15R promoter, control and/or signalsequences may be used, provided such control sequences are compatiblewith the host cell chosen. Exemplary vectors can be constructed asdisclosed by Okayama and Berg, Mol. Cell. Biol. 3:280 (1983).

[0078] A useful system for stable high level expression of mammalianreceptor cDNAs in C127 murine mammary epithelial cells can beconstructed substantially as described by Cosman et al., Mol. Immunol.23:935 (1986).

[0079] In preferred aspects of the present invention, recombinantexpression vectors comprising IL-15R cDNAs are stably integrated into ahost cell's DNA. Elevated levels of expression product are achieved byselecting for cell lines having amplified numbers of vector DNA. Celllines having amplified numbers of vector DNA are selected, for example,by transforming a host cell with a vector comprising a DNA sequence thatencodes an enzyme that is inhibited by a known drug. The vector may alsocomprise a DNA sequence that encodes a desired protein. Alternatively,the host cell may be co-transformed with a second vector that comprisesthe DNA sequence that encodes the desired protein. The transformed orco-transformed host cells are then cultured in increasing concentrationsof the known drug, thereby selecting for drug-resistant cells. Suchdrug-resistant cells survive in increased concentrations of the toxicdrug by over-production of the enzyme that is inhibited by the drug,frequently as a result of amplification of the gene encoding the enzyme.Where drug resistance is caused by an increase in the copy number of thevector DNA encoding the inhibiting enzyme, there is a concomitantco-amplification of the vector DNA encoding the desired protein (e.g.,IL-15R) in the host cell's DNA.

[0080] A preferred system for such co-amplification uses the gene fordihydrofolate reductase (DHFR), that can be inhibited by the drugmethotrexate (MTX). To achieve co-amplification, a host cell that lacksan active gene encoding DHFR is either transformed with a vector thatcomprises DNA sequence encoding DHFR and a desired protein, or isco-transformed with a vector comprising a DNA sequence encoding DHFR anda vector comprising a DNA sequence encoding the desired protein. Thetransformed or co-transformed host cells are cultured in mediacontaining increasing levels of MTX, and those cell lines that surviveare selected.

[0081] A particularly preferred co-amplification system uses the genefor glutamine synthetase (GS), that is responsible for the synthesis ofglutamine from glutamate and ammonia using the hydrolysis of ATP to ADPand phosphate to drive the reaction. GS is subject to inhibition by avariety of inhibitors, for example methionine sulphoximine (MSX). Thus,IL-15R can be expressed in high concentrations by co-amplifying cellstransformed with a vector comprising the DNA sequence for GS and adesired protein, or co-transformed with a vector comprising a DNAsequence encoding GS and a vector comprising a DNA sequence encoding thedesired protein, culturing the host cells in media containing increasinglevels of MSX and selecting for surviving cells. The GS co-amplificationsystem, appropriate recombinant expression vectors and cells lines, aredescribed in the following PCT applications: WO 87/04462, WO 89/01036,WO 89/10404 and WO 86/05807.

[0082] Recombinant proteins are preferably expressed by co-amplificationof DHFR or GS in a mammalian host cell, such as Chinese Hamster Ovary(CHO) cells, or alternatively in a murine myeloma cell line, such asSP2/0-Ag14 or NS0 or a rat myeloma cell line, such as YB2/3.0-Ag20,disclosed in PCT applications WO 89/10404 and WO 86/05807.

[0083] Vectors derived from retroviruses may be employed in mammalianhost cells. A preferred retroviral expression vector is tgLS(+) HyTK,described in PCT application WO 92/08796.

[0084] A preferred eukaryotic vector for expression of IL-15R DNA isdisclosed below in Example 1. This vector, referred to as pDC304, wasderived from pDC302 previously described by Mosley et al., Cell, 59:335-348 (1989) by deleting the adenovirus tripartite leader in pDC302.

[0085] Sense and Antisense Sequences

[0086] The present invention provides both double-stranded andsingle-stranded IL-15R DNA, and IL-15R mRNA as well. The single-strandedIL-15R nucleic acids have use as probes to detect the presence ofhybridizing IL-15R nucleic acids (e.g., in in vitro assays) and as senseand antisense molecules to block expression of IL-15R.

[0087] In one embodiment, the present invention provides antisense orsense molecules comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target IL-15R mRNA (sense) orIL-15R DNA (antisense) sequences. These antisense or sense molecules maycomprise a fragment of the coding region of IL-15R cDNA, and, in oneembodiment, are oligonucleotides comprising at least about 14nucleotides, preferably from about 14 to about 30 nucleotides, of anIL-15R cDNA sequence. The ability to create an antisense or senseoligonucleotide based upon a cDNA sequence for a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659 (1988)and van der Krol et al., BioTechniques 6:958 (1988).

[0088] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that blocktranslation (RNA) or transcription (DNA) by one of several means,including enhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The oligonucleotidesthus may be used to block expression of IL-15R proteins. Uses of theantisense and sense nucleic acid sequences include, but are not limitedto, use as research reagents. The biological effects of blocking IL-15Rexpression in cultured cells may be studied, for example. Theoligonucleotides also may be employed in developing therapeuticprocedures that involve blocking IL-15R expression in vivo.

[0089] Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO 91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are relatively stable invivo (i.e., capable of resisting enzymatic degradation) but retainsequence specificity for binding to target nucleotide sequences. Otherexamples of sense or antisense oligonucleotides include thoseoligonucleotides that are covalently linked to organic moieties such asthose described in WO 90/10448, or to other moieties that increaseaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

[0090] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any suitable method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. A preferred method involves insertion of the antisense or senseoligonucleotide into a suitable retroviral vector, then contacting thetarget cell with the retrovirus vector containing the inserted sequence,either in vivo or ex vivo. Suitable retroviral vectors include, but arenot limited to, the murine retrovirus M-MuLV, N2 (a retrovirus derivedfrom M-MuLV), or the double copy vectors designated DCT5A, DCT5B andDCT5C (see PCT Application US 90/02656).

[0091] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by attaching theoligonucleotide to a molecule that binds to the target cell, asdescribed in WO 91/04753. The oligonucleotide may be attached tomolecules that include, but are not limited to, antibodies, growthfactors, other cytokines, or other ligands that bind to cell surfacereceptors.

[0092] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0093] The following examples are offered by way of illustration, andnot by way of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein, the disclosures of which are incorporated by reference.

EXAMPLES Example 1 Isolation and Expression of cDNAs Encoding MurineIL-15R

[0094] A. Radiolabeling of IL-15. Recombinant flag simian IL-15expressed in yeast (SEQ ID NO:3) was purified by passage over a PhenylSepharose CL-4B column (Pharmacia, Piscataway, N.J.) followed by twopassages over reverse phase HPLC C4 columns (Vydac), the first using apyridine acetate/propanol buffer system, the second in trifluoro-aceticacid acetonitrile system. Fractions containing pure IL-15 were driedunder nitrogen and radiolabeled using the enzymobead iodination reagent(BioRad, Richmond Va.) as described by in Park et al., J. Exp. Med.,165:1201-1206 (1987). The biological activity of radiolabeled IL-15 wasassessed using the mitochondrial stain MTT (3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; thiazol blue(Sigma, St. Dextran, as described by Cosman et al., Nature, 312:768-771(1984)).

[0095] B. Binding To Intact Cells. A source for IL-15R was selected byscreening various murine and human cells lines and tissues forexpression of IL-15R based on their ability to bind ¹²⁵I-IL-15 that wasprepared as described above in Example 1A. For the binding assays, aphthalate oil separation method (Dower et al., J. Immunol. 132:751(1984)) was performed as described by Park et al., J. Biol. Chem261:4177 (1986) and Park et al., Proc. Natl. Acad. Sci. USA 84:5267(1987) on candidate cells grown in suspension culture. Nonspecificbinding of ¹²⁵I-IL-15 was measured in the presence of a 200-fold orgreater molar excess of unlabeled IL-15. Sodium azide (0.2%) wasincluded in all binding assays to inhibit internalization of ¹²⁵I-IL-15at 37° C. Activated PBT and well as antigen specific T cell clonesexpressed only a few hundred receptors for IL-15. Cells from the murineTh2 CD4⁺ cell clone, D10 (Kaye et al., J. Immunol., 133:1339 (1984)),expressed up to 24,000 IL-15 receptors when cultured with IL-2.

[0096] C. Construction and Screening of cDNA Library. PolyadenylatedmRNA was prepared from a D10 cell line and cDNAs were prepared usingstandard techniques. The D10 line is a producer of murine IL-15R. cDNAends were adapted with Bgl II adaptors:

[0097] 5′-GATCTTGGAACGAGACGACCTGCT-3′ (SEQ ID NO:4)

[0098] 3′-AACCTTGCTCTGCTGGACGA-5′ (SEQ ID NO:5)

[0099] and cloned into vector pDC304.

[0100] COS-7 cells were transfected with miniprep DNA from pools of cDNAclones directly on glass slides and cultured for 2-3 days to permittransient expression of IL-15R. The slides containing the transfectedcells were then incubated with medium containing ¹²⁵I-labeled IL-15,washed to remove unbound labeled IL-15, fixed with glutaraldehyde, anddipped in liquid autoradiographic emulsion and exposed in the dark.After developing the slides, they were individually examined with amicroscope and positive cells expressing IL-15R were identified by thepresence of autoradiographic silver grains against a light background.Approximately 20,000 cDNAs were screened in pools of approximately 1000cDNAs each using the slide autoradiographic method until assay of onetransfectant pool showed multiple cells clearly positive for IL-15binding. This pool was then partitioned into pools of approximately 100and again screened by slide autoradiography and a positive pool wasidentified. Individual colonies from this pool of approximately 100 werescreened until a single clone (clone D1-4-D5) was identified thatdirected synthesis of a surface protein with detectable IL-15 bindingactivity. This clone was isolated and sequenced to determine thesequence of the murine IL-15R cDNA clone, D1-4-D5. The cloning vectorpDC304 containing the murine IL-15R cDNA clone, D1-4-D5, was depositedwith the American Type Culture Collection (“ATCC”) under accessionnumber ATCC 62659. The murine IL-15R cDNA insert is made up of a 71-bp5′ noncoding region before an open reading frame of 792 bp and a 995-bp3′ non coding region. The nucleotide sequence of the open reading frameis disclosed in SEQ ID NO:1. The amino acid sequence of a full lengthmurine IL-15R (i.e., including signal peptide, extracellular domain,transmembrane region and cytoplasmic domain) is shown in SEQ ID NOs:1and 2. The amino acid sequence in SEQ ID NOs:1 and 2 predicts a type 1membrane protein (i.e., a single transmembrane region with a N-terminalextracellular domain and a C-terminal cytoplasmic domain). A predictedsignal peptide cleavage occurs between amino acids 30 and 31 in SEQ IDNO:2; amino acids 32 and 33 are predicted to form another, preferred,cleavage site. The predicted transmembrane region includes amino acids206 to 226 in SEQ ID NO:2.

[0101] D. Recombinant IL-15R Binding. Plasmid DNA from IL-15 receptorexpression plasmid was used to transfect a sub-confluent layer of monkeyCOS-7 cells using DEAE-dextran followed by chloroquine treatment, asdescribed by Luthman et al., Nucl Acids Res. 11:1295 (1983) andMcCutchan et al., J. Natl. Cancer Inst. 41:351 (1968). The cells werethen grown in culture for three days to permit transient expression ofthe inserted sequences. After three days, the cell monolayers wereassayed for ¹²⁵1-IL-15 binding essentially as described by Park, et al.,J. Exp. Med. 166:476 (1987). Non-specific binding of ¹²⁵I-L-15 wasmeasured in the presence of 200-fold or greater excess of unlabeledIL-15. Initial binding studies of ¹²⁵I-IL-15 to COS cells transfectedwith IL-15R cDNA clone D1-4-D5 showed very high levels of expression(˜500,000 sites/cell), with an estimated affinity of 1.0-2.2 nM, whichis much lower that the affinity of the native receptor on D10 cells.

[0102] E. Soluble IL-15R. A soluble murine IL-15 receptor was preparedby deleting the transmembrane and cytoplasmic domains, with theC-terminal end corresponding to Thr at amino acid 204 of SEQ ID NO:1,and adding 5 C-terminal Histidines. The soluble IL-15 receptor wasbiologically active, as demonstrated by the fact that it inhibitedbinding of radiolabeled IL-15 to cells expressing membrane bound IL-15receptor (FIG. 1).

Example 2 Preparation of Monoclonal Antibodies to IL-15R

[0103] Preparations of purified recombinant IL-15R, for example, humanIL-15R, or transfected COS cells expressing high levels of IL-15R areemployed to generate monoclonal antibodies against IL-15R usingconventional techniques, for example, those disclosed in U.S. Pat. No.4,411,993. Such antibodies are likely to be useful in interfering withIL-15 binding to IL-15R, for example, in ameliorating undesired effectsof IL-15, or as components of diagnostic or research assays for IL-15 orsoluble IL-15R.

[0104] To immunize mice or rats, IL-15R immunogen is emulsified incomplete Freund's adjuvant and injected in amounts ranging from 10-100μg, subcutaneously. Ten to twenty-one days later, the immunized animalsare boosted with additional immunogen emulsified in incomplete Freund'sadjuvant and periodically boosted thereafter on a weekly to biweeklyimmunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision for testing by dot-blotassay (antibody sandwich) or ELISA (enzyme-linked immunosorbent assay).Other assay procedures are also suitable. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused with an appropriatemurine myeloma cell line. Hybridoma cell lines generated by thisprocedure are plated in multiple microtiter plates in a HAT selectivemedium (hypoxanthine, aminopterin, and thymidine) to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

[0105] Hybridoma clones thus generated can be screened by ELISA forreactivity with IL-15R, for example, by adaptations of the techniquesdisclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Pat.No. 4,703,004. Clones that produce antibodies that bind IL-15R andinhibit binding of IL-15 to IL-15R (blocking or neutralizing antibodies)can also be isolated. Positive clones are then injected into theperitoneal cavities of syngeneic animals to produce ascites containinghigh concentrations (>1 mg/ml) of anti-IL-15R monoclonal antibody. Theresulting monoclonal antibody can be purified by ammonium sulfateprecipitation followed by gel exclusion chromatography, and/or affinitychromatography based on binding of antibody to Protein A ofStaphylococcus aureus.

Example 3 Isolation and Expression of cDNAs Encoding Human IL-15R

[0106] A. Binding of IL-15 to Human Cells

[0107] Various human cell lines and tissues were screened for theability to bind radiolabeled IL-15 substantially as described inExample 1. High affinity binding was observed on activated peripheralblood mononuclear cells, activated monocytes and some EBV-transformedcell lines. High affinity binding was also measured on human fibroblastlines such as W126-VA4 (ATCC CCL 95.1; Kd: 27 pM; 1,400 sites per cell),a glioblastoma cell line, A-172 (ATCC CRL 1620; Kd 50-138 pM;1,560-4,350 sites pre cell), and vascular endothelial cells (both venousand arterial origin, Kd 33 pM, 990 sites per cell; Kd 163 pM, 1,920sites per cell, respectively). Cross-linking of radiolabeled IL-15 toreceptors present on the surface of A172 cells showed a major IL-15binding protein with an estimated molecular weight of 55-65 Kd, a sizesimilar to that seen on the D10 murine cell line by cross-linking.

[0108] B. Clone W5

[0109] A cDNA encoding human IL-15R was isolated by cross-specieshybridization of the murine IL-15R cDNA to a human cDNA library preparedfrom the human cell line W126-VA4-VA4 in the bacteriophage λgt10 vector.Preparation of the library is described in U.S. Pat. No. 5,194,375,issued Mar. 16, 1993 (DNAs Encoding IL-7 Receptors). The W126-VA4library was screened with a random prime labeled murine IL-15R cDNAprobe in 50% formamide buffer (50% formamide, 5×SSC, 20 mM EDTA,2×Denhardt's, 1% SDS, 0.1% sarcosyl, 50 mM KHPO₄ pH 6.5, 300 μg/mlsalmon sperm DNA) using 1×10⁶ cpm of probe/ml of hybridization solution,at 42° C. for 16-20 hours. The filters were washed once with 6×SSC/0. 1%SDS at room temperature, followed by several moderate stringency washesin 2×SSC/0.1% SDS at 55° C.

[0110] Approximately 500,000 plaques of the amplified λgt100/W126-VA4library were screened by standard methods, using the random-primelabeled murine IL-15R probe, which contained the entire coding region aswell as 5′ and 3′ flanking non-coding regions. A single hybridizingplaque was identified, plaque-purified, and its cDNA insert amplified byPCR, purified, and sequenced. This clone, designated ′W5,′ shared about65% identity at the nucleotide level and 56% identity at the amino acidlevel with the murine cDNA, ′D1-4-D5′ (SEQ ID NO: 1). The nucleotide andpredicted amino acid sequence of W5 are shown in SEQ ID NOs:6 and 7.

[0111] As compared to the full-length murine clone D1-4-D5, W5 appearedto be missing a small portion of the expected 5′ sequences, i.e., about125 bp compared to the murine clone, indicating that W5 did not containthe coding region for the first part of a putative IL-15R signal peptide(missing 19 amino acids compared to the murine clone). The amino acidsequence in SEQ ID NOs:6 and 7 predicts a type 1 membrane protein (i.e.,a single transmembrane region with an N-terminal extracellular domainand a C-terminal cytoplasmic domain). The predicted transmembrane regionincludes amino acids 190 to 210 of SEQ ID NOs:6 and 7. Binding of IL-15Rto IL-15 is mediated through the extracellular domain of IL-15R as shownin FIG. 2, all or portions of which are involved in binding.

[0112] A signal peptide cleavage is predicted to occur between aminoacids 14 and 15 in SEQ ID NO:6. For murine IL-15 receptor, a signalpeptide cleavage is predicted to occur between amino acids 32 and 33 inSEQ ID NO:2, or alternatively, between amino acids 30 and 31 in SEQ IDNO:2. Because of the similarity between murine and human (IL-15 receptorin this region, and because the murine IL-7 leader sequence (see below)utilizes a Thr residue as the mature N-terminal amino acid following theleader, the Thr at residue 12 of SEQ ID NOs:6 and 7 was chosen as themature N-terminus of a human IL-15 receptor construct.

[0113] The mature peptide coding domain of W5 (nucleotides 34 through753 of SEQ ID NO:6), and the remaining 3′ non-coding sequence, was fusedto the coding domain for the signal peptide of murine IL-7 in theexpression vector pDC206 (similar to pDC201, described in Sims et al.,Science 241: 585, 1988, with the addition of the murine IL-7 leadersequence, which is described in U.S. Pat. No. 4,965,195, issued Oct. 23,1990). Transfection of this recombinant plasmid into COS-7 cellsfollowed by cell-surface binding of radiolabeled human IL-15substantially as described in Example 1 showed that this plasmid encodeda biologically active polypeptide, i.e., one which bound L-15. The cloneW5 construct containing the murine IL-7 leader sequence in pDC206 wasdeposited with the American Type Culture Collection (“ATCC”, 12301Parklawn Dr., Rockville, Md. 20852, USA), under the conditions of theBudapest Treaty on Sep. 1, 1994, and given accession number ATCC 69690.

[0114] C. Clone P1

[0115] A λgt10 library from human peripheral blood lymphocytes, preparedas described in U.S. Ser. No. 08/094,669, filed Jul. 20, 1993, and inIdzerda et al., J. Exp. Med. 171:861 (1990), was screened for thepresence of a full-length clone encoding human IL-15R using a randomprime labeled human IL15R cDNA probe consisting of the entire W5 cDNAwithout the poly-A tail (which had been removed by digestion of the cDNAwith Ssp I followed by gel purification of the remaining fragment,resulting in a fragment of approximately 1465 bp), using substantiallythe same conditions as described for screening of the A172 library(described below). The resulting sequence of the cDNA insert from thisclone (SEQ ID NOs:8 and 9) exhibited an in-frame insertion of 153basepairs at the mature N-terminus (amino acids 24 through 74 of SEQ IDNOs:8 and 9), an in-frame deletion of 99 basepairs downstream of themature N-terminus that deleted nucleotides 236 through 334 of SEQ IDNO:6 (the sequence encoding amino acids 79 though 112, with thesubstitution of a Lys residue encoded by the codon AAG, the equivalentof nucleotides 235, 335 and 336 of SEQ ID NO:6), and also containedadditional 5′ sequence as compared to clone W5 (amino acids 1 though 10of SEQ ID NOs:8 and 9), but still did not contain an initiator Met.

[0116] D. Clone A212

[0117] A library prepared from A172 cells as described in U.S. Ser. No.08/265,086, filed Jun. 17, 1994, was screened for the presence of afull-length clone encoding human IL-15R. DNA (1-5 μg) from library pools(approximately 1000 cDNA clones/pool) was digested with Sal I to releasethe inserted DNA, electrophoresed (1% agarose, Tris-borate gel), andblotted to a nylon membrane. The blot was probed with a random primelabeled human IL15R cDNA probe consisting of the entire W5 cDNA minusthe poly A tail, under conditions of high stringency (50% formamide, 42°C. hybridization for 16-20 hr, followed by washing at 2×SSC at roomtemperature for 5 minutes followed by 0.1×SSC/0.1% SDS at 68° C.). Theblot was autoradiographed, and a pool with a positive signal (i.e.hybridizing band) was chosen for isolation of individual clones bycolony hybridization.

[0118] A portion of the frozen glycerol stock of the pool of cDNA clonescorresponding to the Southern blot signal was diluted and plated ontoappropriate plates (LB+ampicillin). Colonies were lifted onto nylonmembranes and the membranes processed using standard techniques. Themembranes were hybridized and washed under stringent conditions asdescribed above, and a colony corresponding to a positive hybridizingsignal was grown, its plasmid DNA purified and sequenced. The resultingsequence of the cDNA insert from this clone (SEQ ID NOs: 10 and 11)exhibited the same in-frame deletion of 99 basepairs downstream of themature N-terminus as clone P1 (a deletion of nucleotides 236 through 34of SEQ ID NO:6, the sequence encoding amino acids 79 though 112, withthe substitution of a Lys residue encoded by the codon AAG, theequivalent of nucleotides 235, 335 and 336 of SEQ ID NO:6). The plasmidwas transfected into COS cells, and the ability of the encoded proteinto bind IL-15 determined using slide autoradiography with ¹²⁵1-labeledhuman IL-15 substantially as described in Example 1. Clone A212 alsoencoded a biologically active polypeptide, i.e., one which bound IL-15.Additionally, clone A212 exhibited a complete signal peptide as comparedto clone W5, as indicated by the presence of additional 5′ sequence andan initiator Met.

[0119] E. Clone A133

[0120] A second clone was isolated from the A172 library describedabove, under substantially the same conditions. The nucleotide and aminoacid sequence of the A133 clone are shown in SEQ ID NOs:12 and 13. Thisclone exhibited an incomplete 5′ region which began at the equivalent ofnucleotide 355 of clone W5 (SEQ ID NO:6), and an in-frame insertiondownstream of the transmembrane region that results in a differentcytoplasmic tail coding domain (amino acids 97 through 117 of SEQ IDNOs:12 and 13). A hybrid construct encoding the 5′ half of W5 fused toA133 to give the alternate cytoplasmic tail (SEQ ID NO: 14) wasprepared, and expressed substantially as described above for clone WS.Cell-surface binding experiments using radiolabeled human IL-15substantially as described in Example 1 showed that this hybridconstruct encoded a polypeptide that bound IL-15.

[0121] SEQ ID NO: 15 presents the predicted amino acid sequence of acomposite human IL-15R containing the signal peptide of clone A212 andthe coding region of clone W5. SEQ ID NO: 15 also contains an Xaa atamino acid 182, wherein Xaa is Asn or Thr. Clones W5 and P1 contain aThr at the equivalent position (W5: amino acid 166 of SEQ ID NOs:6 and7; P1: amino acid 194 of SEQ ID NOs:8 and 9), whereas clones A212 andA133 contain an Asn at the equivalent position (A212: amino acid 149 ofSEQ ID NOs:10 and 11; A133: amino acid 48 of SEQ ID NOs: 13 and 14). TheAsn/Thr substitution does not affect binding of IL-15, as evidenced bythe fact that both clones W5 and A212 encoded a peptide that boundIL-15, and may be due to allelic variation.

Example 4

[0122] Characterization of the Role of the α Subunit of the IL-15R

[0123] A. Functional Role for the α Subunit of the IL-15R on MurineCells

[0124] In initial binding experiments with COS-7 cells transfected withthe murine IL-15Ra cDNA clone D5, in excess of 5×10⁵ receptors/cell weredetected, a level too high to obtain accurate measurements of IL-15binding to these cells. More accurate measurements of the affinity ofIL-15Rα for IL-15 were obtained using the murine IL-3 dependent 32D cellline, which constitutively expresses the IL-2R α and γ_(c) chains, butfailed to respond to IL-15 (Grabstein, et al., 1994, supra) as a modelsystem. 32D cells stably expressing various components of the IL-2 andIL-15 receptors were derived and tested for their ability to proliferatein response to IL-15.

[0125] The original 32D cell line responded to IL-2, but a subline,32D-01, which had lost the ability to respond to IL-2 (presumablybecause it no longer expressed sufficient levels of IL-2Rβ) was used inthese experiments. The murine IL-2Rβ chain was introduced into 32D-01,resulting in a line designated 32Dmβ-S, which had the ability toproliferate in response to IL-2 but not IL-15. No detectable IL-15binding to 32D-01 or 32Dmβ-5 was seen by cytofluorometric analysis,suggesting that the level of IL-15Rα was very low on these cells. Directbinding with ¹²⁵I-IL-15 confirmed this result (see below).

[0126] To test the role of IL-15Rα, 32D-01 cells were transfected withthe IL-15Rα cDNA, which resulted in a line expressing the α chain,32Dm15Rα-102. Although these cells bound high levels of IL-15 asevidenced by both cytofluorometric analysis and radiolabeled IL-15binding, they were unable to proliferate in response to IL-15. The32Dm15Roα-102 cells, like the parental 32D-01, did not expressdetectable levels of IL-2Rβ. A cell line termed 32Dmβm15Rα-3,co-expressing both IL-15Rα and IL-2Rβ (γ_(c) is constitutivelyexpressed) was derived, which was able to proliferate in response toIL-15 and IL-2, with a pattern similar to proliferation of the D10 cellline (from which D1-4-D5 [“DS”] was cloned). This result demonstratesthat the ability of murine cells to respond to simian IL-15 is dependenton the level of IL-15Rα expression and confirms the requirement forIL-2Rβ.

[0127] B. IL-15Rα binds IL-15 with High Affinity

[0128] Preliminary equilibrium binding experiments with ¹²⁵I-simianIL-15 indicated that the IL-15Rα chain alone was binding IL-15 with veryhigh affinity; therefore, the optimal binding conditions necessary toaccurately measure this affinity under equilibrium conditions, as wellas to measure whether a receptor complex containing the β and γ_(c)chains along with the IL-15Rα chain exhibited an enhanced affinity forIL-15, were assessed. The parental 32D-01 cell line expressed an averageof 100+33 IL-15 binding sites per cell, with an affinity (K_(a)) of1.4±0.4×10¹¹ M⁻¹, which is similar to the affinity of IL-2 binding tothe IL-2Rα/β/γ_(c) complex. The 32Dm15Rα-102 cells, transfected with theIL-15Rα chain, exhibited a much higher level of IL-15 binding with thesame very high affinity (average of 15300±3700 sites per cell with aK_(a) of 1.5±0.9×10¹¹ M⁻¹). Given the low expression of IL-2Rβ on thesecells, the majority of these sites must reflect binding to the IL-15Rαchain alone. This suggests that the low amount of IL-15 binding on the32D-01 cells is due to endogenous IL-15Rα.

[0129] The affinity of the receptors on both of these 32D lines is verysimilar to the affinity of the native IL-15R on the D10 cells from whichthe IL-15Rα subunit was cloned (average K_(a) of 1.3±0.5×10¹¹ M⁻¹).Although D10 cells express several hundred copies of IL-2Rβ, inferredfrom the number of high affinity IL-2 binding sites (˜500 sites/cell), asecond component of binding in these cells which might correspond to ahigher affinity α/β or α/β/γ_(c) complex was not detected. This resultwas substantiated by analysis of the 32Dmβm15Rα-3 cells, co-expressingboth recombinant IL-15Rot and IL-2Rβ subunits. These cells showedbinding characteristics very similar to those exhibited by the32Dm15Rα-102 cells, with an average K_(a) of 2.2±0.3×10¹¹ M⁻¹, and12800±2700 receptors/cell.

[0130] In both D10 and 32Dmβm15Rα-3 cells, overexpression of the IL-15Rαrelative to the β subunit might serve to obscure a small higher affinitycomponent. This possibility was addressed by analyzing binding to the32Dmβ-5 cell line, which had been transfected with the β subunit alone.These cells showed a single high affinity binding site that wasessentially identical to the parental 32D-01 line, with an average K_(a)of 1.9±0.5×10¹¹ M⁻¹ and 40±15 sites per cell, presumably due to lowlevel expression of endogenous IL-15Rα. The observation that the 32Dmβ-5cell line did not display any additional IL-15 binding sites relative tothe 32D-01 parent line indicated that simian IL-15 is unable to bindwith any detectable affinity to complexes of murine β and γ_(c), in theabsence of the IL-15Rα chain.

1 15 792 base pairs nucleic acid single linear cDNA NO NO CDS 1..789 1ATG GCC TCG CCG CAG CTC CGG GGC TAT GGA GTC CAG GCC ATT CCT GTG 48 MetAla Ser Pro Gln Leu Arg Gly Tyr Gly Val Gln Ala Ile Pro Val 1 5 10 15TTG CTG CTG CTG CTG TTG CTA CTG TTG CTC CCG CTG AGG GTG ACG CCG 96 LeuLeu Leu Leu Leu Leu Leu Leu Leu Leu Pro Leu Arg Val Thr Pro 20 25 30 GGCACC ACG TGT CCA CCT CCC GTA TCT ATT GAG CAT GCT GAC ATC CGG 144 Gly ThrThr Cys Pro Pro Pro Val Ser Ile Glu His Ala Asp Ile Arg 35 40 45 GTC AAGAAT TAC AGT GTG AAC TCC AGG GAG AGG TAT GTC TGT AAC TCT 192 Val Lys AsnTyr Ser Val Asn Ser Arg Glu Arg Tyr Val Cys Asn Ser 50 55 60 GGC TTT AAGCGG AAA GCT GGA ACA TCC ACC CTG ATT GAG TGT GTG ATC 240 Gly Phe Lys ArgLys Ala Gly Thr Ser Thr Leu Ile Glu Cys Val Ile 65 70 75 80 AAC AAG AACACA AAT GTT GCC CAC TGG ACA ACT CCC AGC CTC AAG TGC 288 Asn Lys Asn ThrAsn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys 85 90 95 ATC AGA GAC CCCTCC CTA GCT CAC TAC AGT CCA GTG CCA ACA GTA GTG 336 Ile Arg Asp Pro SerLeu Ala His Tyr Ser Pro Val Pro Thr Val Val 100 105 110 ACA CCA AAG GTGACC TCA CAG CCA GAG AGC CCC TCC CCC TCT GCA AAA 384 Thr Pro Lys Val ThrSer Gln Pro Glu Ser Pro Ser Pro Ser Ala Lys 115 120 125 GAG CCA GAA GCTTTC TCT CCC AAA TCA GAT ACC GCA ATG ACC ACA GAG 432 Glu Pro Glu Ala PheSer Pro Lys Ser Asp Thr Ala Met Thr Thr Glu 130 135 140 ACA GCT ATT ATGCCT GGC TCC AGG CTG ACA CCA TCC CAA ACA ACT TCT 480 Thr Ala Ile Met ProGly Ser Arg Leu Thr Pro Ser Gln Thr Thr Ser 145 150 155 160 GCA GGA ACTACA GGG ACA GGC AGT CAC AAG TCC TCC CGA GCC CCA TCT 528 Ala Gly Thr ThrGly Thr Gly Ser His Lys Ser Ser Arg Ala Pro Ser 165 170 175 CTT GCA GCAACA ATG ACC TTG GAG CCT ACA GCC TCC ACC TCC CTC AGG 576 Leu Ala Ala ThrMet Thr Leu Glu Pro Thr Ala Ser Thr Ser Leu Arg 180 185 190 ATA ACA GAGATT TCT CCC CAC AGT TCC AAA ATG ACG AAA GTG GCC ATC 624 Ile Thr Glu IleSer Pro His Ser Ser Lys Met Thr Lys Val Ala Ile 195 200 205 TCT ACA TCGGTC CTC TTG GTT GGT GCA GGG GTT GTG ATG GCT TTC CTG 672 Ser Thr Ser ValLeu Leu Val Gly Ala Gly Val Val Met Ala Phe Leu 210 215 220 GCC TGG TACATC AAA TCA AGG CAG CCT TCT CAG CCG TGC CGT GTT GAG 720 Ala Trp Tyr IleLys Ser Arg Gln Pro Ser Gln Pro Cys Arg Val Glu 225 230 235 240 GTG GAAACC ATG GAA ACA GTA CCA ATG ACT GTG AGG GCC AGC AGC AAG 768 Val Glu ThrMet Glu Thr Val Pro Met Thr Val Arg Ala Ser Ser Lys 245 250 255 GAG GATGAA GAC ACA GGA GCC TAA 792 Glu Asp Glu Asp Thr Gly Ala 260 263 aminoacids amino acid linear protein 2 Met Ala Ser Pro Gln Leu Arg Gly TyrGly Val Gln Ala Ile Pro Val 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu LeuLeu Leu Pro Leu Arg Val Thr Pro 20 25 30 Gly Thr Thr Cys Pro Pro Pro ValSer Ile Glu His Ala Asp Ile Arg 35 40 45 Val Lys Asn Tyr Ser Val Asn SerArg Glu Arg Tyr Val Cys Asn Ser 50 55 60 Gly Phe Lys Arg Lys Ala Gly ThrSer Thr Leu Ile Glu Cys Val Ile 65 70 75 80 Asn Lys Asn Thr Asn Val AlaHis Trp Thr Thr Pro Ser Leu Lys Cys 85 90 95 Ile Arg Asp Pro Ser Leu AlaHis Tyr Ser Pro Val Pro Thr Val Val 100 105 110 Thr Pro Lys Val Thr SerGln Pro Glu Ser Pro Ser Pro Ser Ala Lys 115 120 125 Glu Pro Glu Ala PheSer Pro Lys Ser Asp Thr Ala Met Thr Thr Glu 130 135 140 Thr Ala Ile MetPro Gly Ser Arg Leu Thr Pro Ser Gln Thr Thr Ser 145 150 155 160 Ala GlyThr Thr Gly Thr Gly Ser His Lys Ser Ser Arg Ala Pro Ser 165 170 175 LeuAla Ala Thr Met Thr Leu Glu Pro Thr Ala Ser Thr Ser Leu Arg 180 185 190Ile Thr Glu Ile Ser Pro His Ser Ser Lys Met Thr Lys Val Ala Ile 195 200205 Ser Thr Ser Val Leu Leu Val Gly Ala Gly Val Val Met Ala Phe Leu 210215 220 Ala Trp Tyr Ile Lys Ser Arg Gln Pro Ser Gln Pro Cys Arg Val Glu225 230 235 240 Val Glu Thr Met Glu Thr Val Pro Met Thr Val Arg Ala SerSer Lys 245 250 255 Glu Asp Glu Asp Thr Gly Ala 260 122 amino acidsamino acid linear protein NO 3 Asp Tyr Lys Asp Asp Asp Asp Lys Asn TrpVal Asn Val Ile Ser Asp 1 5 10 15 Leu Lys Lys Ile Glu Asp Leu Ile GlnSer Met His Ile Asp Ala Thr 20 25 30 Leu Tyr Thr Glu Ser Asp Val His ProSer Cys Lys Val Thr Ala Met 35 40 45 Lys Cys Phe Leu Leu Glu Leu Gln ValIle Ser His Glu Ser Gly Asp 50 55 60 Thr Asp Ile His Asp Thr Val Glu AsnLeu Ile Ile Leu Ala Asn Asn 65 70 75 80 Ile Leu Ser Ser Asn Gly Asn IleThr Glu Ser Gly Cys Lys Glu Cys 85 90 95 Glu Glu Leu Glu Glu Lys Asn IleLys Glu Phe Leu Gln Ser Phe Val 100 105 110 His Ile Val Gln Met Phe IleAsn Thr Ser 115 120 24 base pairs nucleic acid single linear cDNA NO NO4 GATCTTGGAA CGAGACGACC TGCT 24 20 base pairs nucleic acid single linearcDNA NO NO 5 AGCAGGTCGT CTCGTTCCAA 20 1534 base pairs nucleic acidsingle linear cDNA NO NO CDS 1..753 6 CTG CTA CTG CTG CTG CTG CTC CGGCCG CCG GCG ACG CGG GGC ATC ACG 48 Leu Leu Leu Leu Leu Leu Leu Arg ProPro Ala Thr Arg Gly Ile Thr 1 5 10 15 TGC CCT CCC CCC ATG TCC GTG GAACAC GCA GAC ATC TGG GTC AAG AGC 96 Cys Pro Pro Pro Met Ser Val Glu HisAla Asp Ile Trp Val Lys Ser 20 25 30 TAC AGC TTG TAC TCC AGG GAG CGG TACATT TGT AAC TCT GGT TTC AAG 144 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr IleCys Asn Ser Gly Phe Lys 35 40 45 CGT AAA GCC GGC ACG TCC AGC CTG ACG GAGTGC GTG TTG AAC AAG GCC 192 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu CysVal Leu Asn Lys Ala 50 55 60 ACG AAT GTC GCC CAC TGG ACA ACC CCC AGT CTCAAA TGC ATT AGA GAC 240 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu LysCys Ile Arg Asp 65 70 75 80 CCT GCC CTG GTT CAC CAA AGG CCA GCG CCA CCCTCC ACA GTA ACG ACG 288 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro SerThr Val Thr Thr 85 90 95 GCA GGG GTG ACC CCA CAG CCA GAG AGC CTC TCC CCTTCT GGA AAA GAG 336 Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro SerGly Lys Glu 100 105 110 CCC GCA GCT TCA TCT CCC AGC TCA AAC AAC ACA GCGGCC ACA ACA GCA 384 Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala AlaThr Thr Ala 115 120 125 GCT ATT GTC CCG GGC TCC CAG CTG ATG CCT TCA AAATCA CCT TCC ACA 432 Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys SerPro Ser Thr 130 135 140 GGA ACC ACA GAG ATA AGC AGT CAT GAG TCC TCC CACGGC ACC CCC TCT 480 Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His GlyThr Pro Ser 145 150 155 160 CAG ACA ACA GCC AAG ACC TGG GAA CTC ACA GCATCC GCC TCC CAC CAG 528 Gln Thr Thr Ala Lys Thr Trp Glu Leu Thr Ala SerAla Ser His Gln 165 170 175 CCG CCA GGT GTG TAT CCA CAG GGC CAC AGC GACACC ACT GTG GCT ATC 576 Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp ThrThr Val Ala Ile 180 185 190 TCC ACG TCC ACT GTC CTG CTG TGT GGG CTG AGCGCT GTG TCT CTC CTG 624 Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser AlaVal Ser Leu Leu 195 200 205 GCA TGC TAC CTC AAG TCA AGG CAA ACT CCC CCGCTG GCC AGC GTT GAA 672 Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro LeuAla Ser Val Glu 210 215 220 ATG GAA GCC ATG GAG GCT CTG CCG GTG ACT TGGGGG ACC AGC AGC AGA 720 Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp GlyThr Ser Ser Arg 225 230 235 240 GAT GAA GAC TTG GAA AAC TGC TCT CAC CACCTA TGAAACTCGG GGAAACCAGC 773 Asp Glu Asp Leu Glu Asn Cys Ser His HisLeu 245 250 CCAGCTAAGT CCGGAGTGAA GGAGCCTCTC TGCTTTAGCT AAAGACGACTGAGAAGAGGT 833 GCAAGGAAGC GGGCTCCAGG AGCAAGCTCA CCAGGCCTCT CAGAAGTCCCAGCAGGATCT 893 CACGGACTGC CGGGTCGGCG CCTCCTGCGC GAGGGAGCAG GTTCTCCGCATTCCCATGGG 953 CACCACCTGC CTGCCTGTCG TGCCTTGGAC CCAGGGCCCA GCTTCCCAGGAGAGACCAAA 1013 GGCTTCTGAG CAGGATTTTT ATTTCATTAC AGTGTGAGCT GCCTGGAATACATGTGGTAA 1073 TGAAATAAAA ACCCTGCCCC GAATCTTCCG TCCCTCATCC TAACTTGCAGTTCACAGAGA 1133 AAAGTGACAT ACCCAAAGCT CTCTGTCAAT TACAAGGCTT CTCCTGGCGTGGGAGACGTC 1193 TACAGGGAAG ACACCAGCGT TTGGGCTTCT AACCACCCTG TCTCCAGCTGCTCTGCACAC 1253 ATGGACAGGG ACCTGGGAAA GGTGGGAGAG ATGCTGAGCC CAGCGAATCCTCTCCATTGA 1313 AGGATTCAGG AAGAAGAAAA CTCAACTCAG TGCCATTTTA CGAATATATGCGTTTATATT 1373 TATACTTCCT TGTCTATTAT ATCTATACAT TATATATTAT TTGTATTTTGACATTGTACC 1433 TTGTATAAAC AAAATAAAAC ATCTATTTTC AATATTTTTA AAATGCAAAAAAAAAAAAAA 1493 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A 1534 251amino acids amino acid linear protein 7 Leu Leu Leu Leu Leu Leu Leu ArgPro Pro Ala Thr Arg Gly Ile Thr 1 5 10 15 Cys Pro Pro Pro Met Ser ValGlu His Ala Asp Ile Trp Val Lys Ser 20 25 30 Tyr Ser Leu Tyr Ser Arg GluArg Tyr Ile Cys Asn Ser Gly Phe Lys 35 40 45 Arg Lys Ala Gly Thr Ser SerLeu Thr Glu Cys Val Leu Asn Lys Ala 50 55 60 Thr Asn Val Ala His Trp ThrThr Pro Ser Leu Lys Cys Ile Arg Asp 65 70 75 80 Pro Ala Leu Val His GlnArg Pro Ala Pro Pro Ser Thr Val Thr Thr 85 90 95 Ala Gly Val Thr Pro GlnPro Glu Ser Leu Ser Pro Ser Gly Lys Glu 100 105 110 Pro Ala Ala Ser SerPro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala 115 120 125 Ala Ile Val ProGly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr 130 135 140 Gly Thr ThrGlu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser 145 150 155 160 GlnThr Thr Ala Lys Thr Trp Glu Leu Thr Ala Ser Ala Ser His Gln 165 170 175Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile 180 185190 Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu 195200 205 Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu210 215 220 Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser SerArg 225 230 235 240 Asp Glu Asp Leu Glu Asn Cys Ser His His Leu 245 2501641 base pairs nucleic acid single linear cDNA NO NO CDS 3..839 8 CGCGC GGC TGC CGG ACC CTC GGT CTC CCG GCG CTG CTA CTG CTG CTG 47 Arg GlyCys Arg Thr Leu Gly Leu Pro Ala Leu Leu Leu Leu Leu 1 5 10 15 CTG CTCCGG CCG CCG GCG ACG CGG GAT GCA AGA GAC AGG CTG GCT GTC 95 Leu Leu ArgPro Pro Ala Thr Arg Asp Ala Arg Asp Arg Leu Ala Val 20 25 30 CTG GCG GGAAGG AGC AGA ATA TCT GAA AGC TTC AAC CAT GAG GTC CAG 143 Leu Ala Gly ArgSer Arg Ile Ser Glu Ser Phe Asn His Glu Val Gln 35 40 45 ACA CAC GAG GCCTGC GTG AGA CTC AGG ACA ATG GAA AAC TGC CCC CAG 191 Thr His Glu Ala CysVal Arg Leu Arg Thr Met Glu Asn Cys Pro Gln 50 55 60 TGC CAC CAC CAT CGGACA AGC AGG CAG CAA GCA GGC ATC ACG TGC CCT 239 Cys His His His Arg ThrSer Arg Gln Gln Ala Gly Ile Thr Cys Pro 65 70 75 CCC CCC ATG TCC GTG GAACAC GCA GAC ATC TGG GTC AAG AGC TAC AGC 287 Pro Pro Met Ser Val Glu HisAla Asp Ile Trp Val Lys Ser Tyr Ser 80 85 90 95 TTG TAC TCC AGG GAG CGGTAC ATT TGT AAC TCT GGT TTC AAG CGT AAA 335 Leu Tyr Ser Arg Glu Arg TyrIle Cys Asn Ser Gly Phe Lys Arg Lys 100 105 110 GCC GGC ACG TCC AGC CTGACG GAG TGC GTG TTG AAC AAG GCC ACG AAT 383 Ala Gly Thr Ser Ser Leu ThrGlu Cys Val Leu Asn Lys Ala Thr Asn 115 120 125 GTC GCC CAC TGG ACA ACCCCC AGT CTC AAA TGC ATT AAG CCC GCA GCT 431 Val Ala His Trp Thr Thr ProSer Leu Lys Cys Ile Lys Pro Ala Ala 130 135 140 TCA TCT CCC AGC TCA AACAAC ACA GCG GCC ACA ACA GCA GCT ATT GTC 479 Ser Ser Pro Ser Ser Asn AsnThr Ala Ala Thr Thr Ala Ala Ile Val 145 150 155 CCG GGC TCC CAG CTG ATGCCT TCA AAA TCA CCT TCC ACA GGA ACC ACA 527 Pro Gly Ser Gln Leu Met ProSer Lys Ser Pro Ser Thr Gly Thr Thr 160 165 170 175 GAG ATA AGC AGT CATGAG TCC TCC CAC GGC ACC CCC TCT CAG ACA ACA 575 Glu Ile Ser Ser His GluSer Ser His Gly Thr Pro Ser Gln Thr Thr 180 185 190 GCC AAG ACC TGG GAACTC ACA GCA TCC GCC TCC CAC CAG CCG CCA GGT 623 Ala Lys Thr Trp Glu LeuThr Ala Ser Ala Ser His Gln Pro Pro Gly 195 200 205 GTG TAT CCA CAG GGCCAC AGC GAC ACC ACT GTG GCT ATC TCC ACG TCC 671 Val Tyr Pro Gln Gly HisSer Asp Thr Thr Val Ala Ile Ser Thr Ser 210 215 220 ACT GTC CTG CTG TGTGGG CTG AGC GCT GTG TCT CTC CTG GCA TGC TAC 719 Thr Val Leu Leu Cys GlyLeu Ser Ala Val Ser Leu Leu Ala Cys Tyr 225 230 235 CTC AAG TCA AGG CAAACT CCC CCG CTG GCC AGC GTT GAA ATG GAA GCC 767 Leu Lys Ser Arg Gln ThrPro Pro Leu Ala Ser Val Glu Met Glu Ala 240 245 250 255 ATG GAG GCT CTGCCG GTG ACT TGG GGG ACC AGC AGC AGA GAT GAA GAC 815 Met Glu Ala Leu ProVal Thr Trp Gly Thr Ser Ser Arg Asp Glu Asp 260 265 270 TTG GAA AAC TGCTCT CAC CAC CTA TGAAACTCAG GGAAACCAGC CCAGCTAAGT 869 Leu Glu Asn Cys SerHis His Leu 275 CCGGAGTGAA GGAGCCTCTC TGCTTTAGCT AAAGACGACT GAGAAGAGGTGCAAGGAAGC 929 GGGCTCCAGG AGCAAGCTCA CCAGGCCTCT CAGAAGTCCC AGCAGGATCTCACGGACTGC 989 CGGGTCGGCG CCTCCTGCGC GAGGGAGCAG GTTCTCCGCA TTCCCATGGGCACCACCTGC 1049 CTGCCTGTCG TGCCTTGGAC CCAGGGCCCA GCTTCCCAGG AGAGACCAAAGGCTTCTGAG 1109 CAGGATTTTT ATTTCATTAC AGTGTGAGCT GCCTGGAATA CATGTGGTAATGAAATAAAA 1169 ACCCTGCCCC GAATCTTCCG TCCCTCATCC TAACTTGCAG TTCACAGAGAAAAGTGACAT 1229 ACCCAAAGCT CTCTGTCAAT TACAAGGCTT CTCCTGGCGT GGGAGACGTCTACAGGGAAG 1289 ACACCAGCGT TTGGGCTTCT AACCACCCTG TCTCCAGCTG CTCTGCACACATGGACAGGG 1349 ACCTGGGAAA GGTGGGAGAG ATGCTGAGCC CAGCGAATCC TCTCCATTGAAGGATTCAGG 1409 AAGAAGAAAA CTCAACTCAG TGCCATTTTA CGAATATATG CGTTTATATTTATACTTCCT 1469 TGTCTATTAT ATCTATACAT TATATATTAT TTGTATTTTG ACATTGTACCTTGTATAAAC 1529 AAAATAAAAC ATCTATTTTC AATAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA 1589 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAA 1641 279 amino acids amino acid linear protein 9 Arg Gly Cys Arg ThrLeu Gly Leu Pro Ala Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Arg Pro ProAla Thr Arg Asp Ala Arg Asp Arg Leu Ala Val Leu 20 25 30 Ala Gly Arg SerArg Ile Ser Glu Ser Phe Asn His Glu Val Gln Thr 35 40 45 His Glu Ala CysVal Arg Leu Arg Thr Met Glu Asn Cys Pro Gln Cys 50 55 60 His His His ArgThr Ser Arg Gln Gln Ala Gly Ile Thr Cys Pro Pro 65 70 75 80 Pro Met SerVal Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu 85 90 95 Tyr Ser ArgGlu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala 100 105 110 Gly ThrSer Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val 115 120 125 AlaHis Trp Thr Thr Pro Ser Leu Lys Cys Ile Lys Pro Ala Ala Ser 130 135 140Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala Ala Ile Val Pro 145 150155 160 Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr Gly Thr Thr Glu165 170 175 Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser Gln Thr ThrAla 180 185 190 Lys Thr Trp Glu Leu Thr Ala Ser Ala Ser His Gln Pro ProGly Val 195 200 205 Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile SerThr Ser Thr 210 215 220 Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu LeuAla Cys Tyr Leu 225 230 235 240 Lys Ser Arg Gln Thr Pro Pro Leu Ala SerVal Glu Met Glu Ala Met 245 250 255 Glu Ala Leu Pro Val Thr Trp Gly ThrSer Ser Arg Asp Glu Asp Leu 260 265 270 Glu Asn Cys Ser His His Leu 2751474 base pairs nucleic acid single linear cDNA NO NO CDS 83..784 10CCCAGAGCAG CGCTCGCCAC CTCCCCCCGG CCTGGGCAGC GCTCGCCCGG GGAGTCCAGC 60GGTGTCCTGT GGAGCTGCCG CC ATG GCC CCG CGG CGG GCG CGC GGC TGC CGG 112 MetAla Pro Arg Arg Ala Arg Gly Cys Arg 1 5 10 ACC CTC GGT CTC CCG GCG CTGCTA CTG CTG CTG CTG CTC CGG CCG CCG 160 Thr Leu Gly Leu Pro Ala Leu LeuLeu Leu Leu Leu Leu Arg Pro Pro 15 20 25 GCG ACG CGG GGC ATC ACG TGC CCTCCC CCC ATG TCC GTG GAA CAC GCA 208 Ala Thr Arg Gly Ile Thr Cys Pro ProPro Met Ser Val Glu His Ala 30 35 40 GAC ATC TGG GTC AAG AGC TAC AGC TTGTAC TCC AGG GAG CGG TAC ATT 256 Asp Ile Trp Val Lys Ser Tyr Ser Leu TyrSer Arg Glu Arg Tyr Ile 45 50 55 TGT AAC TCT GGT TTC AAG CGT AAA GCC GGCACG TCC AGC CTG ACG GAG 304 Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly ThrSer Ser Leu Thr Glu 60 65 70 TGC GTG TTG AAC AAG GCC ACG AAT GTC GCC CACTGG ACA ACC CCC AGT 352 Cys Val Leu Asn Lys Ala Thr Asn Val Ala His TrpThr Thr Pro Ser 75 80 85 90 CTC AAA TGC ATT AAG CCC GCA GCT TCA TCT CCCAGC TCA AAC AAC ACA 400 Leu Lys Cys Ile Lys Pro Ala Ala Ser Ser Pro SerSer Asn Asn Thr 95 100 105 GCG GCC ACA ACA GCA GCT ATT GTC CCG GGC TCCCAG CTG ATG CCT TCA 448 Ala Ala Thr Thr Ala Ala Ile Val Pro Gly Ser GlnLeu Met Pro Ser 110 115 120 AAA TCA CCT TCC ACA GGA ACC ACA GAG ATA AGCAGT CAT GAG TCC TCC 496 Lys Ser Pro Ser Thr Gly Thr Thr Glu Ile Ser SerHis Glu Ser Ser 125 130 135 CAC GGC ACC CCC TCT CAG ACA ACA GCC AAG AACTGG GAA CTC ACA GCA 544 His Gly Thr Pro Ser Gln Thr Thr Ala Lys Asn TrpGlu Leu Thr Ala 140 145 150 TCC GCC TCC CAC CAG CCG CCA GGT GTG TAT CCACAG GGC CAC AGC GAC 592 Ser Ala Ser His Gln Pro Pro Gly Val Tyr Pro GlnGly His Ser Asp 155 160 165 170 ACC ACT GTG GCT ATC TCC ACG TCC ACT GTCCTG CTG TGT GGG CTG AGC 640 Thr Thr Val Ala Ile Ser Thr Ser Thr Val LeuLeu Cys Gly Leu Ser 175 180 185 GCT GTG TCT CTC CTG GCA TGC TAC CTC AAGTCA AGG CAA ACT CCC CCG 688 Ala Val Ser Leu Leu Ala Cys Tyr Leu Lys SerArg Gln Thr Pro Pro 190 195 200 CTG GCC AGC GTT GAA ATG GAA GCC ATG GAGGCT CTG CCG GTG ACT TGG 736 Leu Ala Ser Val Glu Met Glu Ala Met Glu AlaLeu Pro Val Thr Trp 205 210 215 GGG ACC AGC AGC AGA GAT GAA GAC TTG GAAAAC TGC TCT CAC CAC CTA 784 Gly Thr Ser Ser Arg Asp Glu Asp Leu Glu AsnCys Ser His His Leu 220 225 230 TGAAACTCGG GGAAACCAGC CCAGCTAAGTCCGGAGTGAA GGAGCCTCTC TGCTTTAGCT 844 AAAGACGACT GAGAAGAGGT GCAAGGAAGCGGGCTCCAGG AGCAAGCTCA CCAGGCCTCT 904 CAGAAGTCCC AGCAGGATCT CACGGACTGCCGGGTCGGCG CCTCCTGCGC GAGGGAGCAG 964 GTTCTCCGCA TTCCCATGGG CACCACCTGCCTGCCTGTCG TGCCTTGGAC CCAGGGCCCA 1024 GCTTCCCAGG AGAGACCAAA GGCTTCTGAGCAGGATTTTT ATTTCATTAC AGTGTGAGCT 1084 GCCTGGAATA CATGTGGTAA TGAAATAAAAACCCTGCCCC GAATCTTCCG TCCCTCATCC 1144 TAACTTTCAG TTCACAGAGA AAAGTGACATACCCAAAGCT CTCTGTCAAT TACAAGGCTT 1204 CTCCTGGCGT GGGAGACGTC TACAGGGAAGACACCAGCGT TTGGGCTTCT AACCACCCTG 1264 TCTCCAGCTG CTCTGCACAC ATGGACAGGGACCTGGGAAA GGTGGGAGAG ATGCTGAGCC 1324 CAGCGAATCC TCTCCATTGA AGGATTCAGGAAGAAGAAAA CTCAACTCAG TGCCATTTTA 1384 CGAATATATG CGTTTATATT TATACTTCCTTGTCTATTAT ATCTATACAT TATATATTAT 1444 TTGTATTTTG ACATTGTACC TTGTATAAAC1474 234 amino acids amino acid linear protein 11 Met Ala Pro Arg ArgAla Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala 1 5 10 15 Leu Leu Leu LeuLeu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr 20 25 30 Cys Pro Pro ProMet Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 35 40 45 Tyr Ser Leu TyrSer Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 50 55 60 Arg Lys Ala GlyThr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 65 70 75 80 Thr Asn ValAla His Trp Thr Thr Pro Ser Leu Lys Cys Ile Lys Pro 85 90 95 Ala Ala SerSer Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala Ala 100 105 110 Ile ValPro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr Gly 115 120 125 ThrThr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser Gln 130 135 140Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln Pro 145 150155 160 Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile Ser165 170 175 Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu LeuAla 180 185 190 Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser ValGlu Met 195 200 205 Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr SerSer Arg Asp 210 215 220 Glu Asp Leu Glu Asn Cys Ser His His Leu 225 2301510 base pairs nucleic acid single linear cDNA NO NO CDS 3..356 12 CCAGC TCA AAC AAC ACA GCG GCC ACA ACA GCA GCT ATT GTC CCG GGC 47 Ser SerAsn Asn Thr Ala Ala Thr Thr Ala Ala Ile Val Pro Gly 1 5 10 15 TCC CAGCTG ATG CCT TCA AAA TCA CCT TCC ACA GGA ACC ACA GAG ATA 95 Ser Gln LeuMet Pro Ser Lys Ser Pro Ser Thr Gly Thr Thr Glu Ile 20 25 30 AGC AGT CATGAG TCC TCC CAC GGC ACC CCC TCT CAG ACA ACA GCC AAG 143 Ser Ser His GluSer Ser His Gly Thr Pro Ser Gln Thr Thr Ala Lys 35 40 45 AAC TGG GAA CTCACA GCA TCC GCC TCC CAC CAG CCG CCA GGT GTG TAT 191 Asn Trp Glu Leu ThrAla Ser Ala Ser His Gln Pro Pro Gly Val Tyr 50 55 60 CCA CAG GGC CAC AGCGAC ACC ACT GTG GCT ATC TCC ACG TCC ACT GTC 239 Pro Gln Gly His Ser AspThr Thr Val Ala Ile Ser Thr Ser Thr Val 65 70 75 CTG CTG TGT GGG CTG AGCGCT GTG TCT CTC CTG GCA TGC TAC CTC AAG 287 Leu Leu Cys Gly Leu Ser AlaVal Ser Leu Leu Ala Cys Tyr Leu Lys 80 85 90 95 TCA AGG GCC TCT GTC TGCTCC TGC CAT CCC CGC AGT GCT GGA CAT ACA 335 Ser Arg Ala Ser Val Cys SerCys His Pro Arg Ser Ala Gly His Thr 100 105 110 TGC TCA GTG GGA AGC GTCTGT TGATTTGAGG GCAACCCCCT CCTCTTTTCA 386 Cys Ser Val Gly Ser Val Cys 115AAACCTATGA ACCACCTGCT TTGCAGGCAA ACTCCCCCGC TGGCCAGCGT TGAAATGGAA 446GCCATGGAGG CTCTGCCGGT GACTTGGGGG ACCAGCAGCA GAGATGAAGA CTTGGAAAAC 506TGCTCTCACC ACCTATGAAA CTCGGGGAAA CCAGCCCAGC TAAGTCCGGA GTGAAGGAGC 566CTCTCTGCTT TAGCTAAAGA CGACTGAGAA GAGGTGCAAG GAAGCGGGCT CCAGGAGCAA 626GCTCACCAGG CCTCTCAGAA GTCCCAGCAG GATCTCACGG ACTGCCGGGT CGGCGCCTCC 686TGCGCGAGGG AGCAGGTTCT CCGCATTCCC ATGGGCACCA CCTGCCTGCC TGTCGTGCCT 746TGGACCCAGG GCCCAGCTTC CCAGGAGAGA CCAAAGGCTT CTGAGCAGGA TTTTTATTTC 806ATTACAGTGT GAGCTGCCTG GAATACATGT GGTAATGAAA TAAAAACCCT GCCCCGAATC 866TTCCGTCCCT CATCCTAACT TTCAGTTCAC AGAGAAAAGT GACATACCCA AAGCTCTCTG 926TCAATTACAA GGCTTCTCCT GGCCTGGGAG ACGTCTACAG GGAAGACACC AGCGTTTGGG 986CTTCTAACCA CCCTGTCTCC AGCTGCTCTG CACACATGGA CAGGGACCTG GGAAAGGTGG 1046GAGAGATGCT GAGCCCAGCG AATCCTCTCC ATTGAAGGAT TCAGGAAGAA GAAAACTCAA 1106CTCAGTGCCA TTTTACGAAT ATATGCGTTT ATATTTATAC TTCCTTGTCT ATTATATCTA 1166TACATTATAT ATTATTTGTA TTTTGACATT GTACCTTGTA TAAACAAAAT AAAACATCTA 1226TTTTCAATAT TTTTAAAATG CATTAAGAGA ATCACCAAGG AGAAATGTTC CACATAAAGG 1286AGGAGAAAGA GTAGGAAGGC AGAGTCCAAG GTGACTGAGT TCAGGTGTTC TTTCCAGAAG 1346GAGAAAAAGC CTTGCCTAAA GCTGGCTCCG GTCACAGTTT TGGGGAATTT CCCACAATTC 1406CATGTGAGGA GAAGCAGCAT TATCTAATCC ACACAGTGGC AAGTCTGGGC TCAGCTCCCC 1466AGTGGTATAC ACATCGTCTC TTCCCTTCTT CTTCTCTTAC TTTC 1510 118 amino acidsamino acid linear protein 13 Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala AlaIle Val Pro Gly Ser 1 5 10 15 Gln Leu Met Pro Ser Lys Ser Pro Ser ThrGly Thr Thr Glu Ile Ser 20 25 30 Ser His Glu Ser Ser His Gly Thr Pro SerGln Thr Thr Ala Lys Asn 35 40 45 Trp Glu Leu Thr Ala Ser Ala Ser His GlnPro Pro Gly Val Tyr Pro 50 55 60 Gln Gly His Ser Asp Thr Thr Val Ala IleSer Thr Ser Thr Val Leu 65 70 75 80 Leu Cys Gly Leu Ser Ala Val Ser LeuLeu Ala Cys Tyr Leu Lys Ser 85 90 95 Arg Ala Ser Val Cys Ser Cys His ProArg Ser Ala Gly His Thr Cys 100 105 110 Ser Val Gly Ser Val Cys 115 225amino acids amino acid single linear Protein YES NO 14 Thr Arg Gly IleThr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp 1 5 10 15 Ile Trp ValLys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys 20 25 30 Asn Ser GlyPhe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys 35 40 45 Val Leu AsnLys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu 50 55 60 Lys Cys IleArg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro 65 70 75 80 Ser ThrVal Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser 85 90 95 Pro SerGly Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr 100 105 110 AlaAla Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser 115 120 125Lys Ser Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser 130 135140 His Gly Thr Pro Ser Gln Thr Thr Ala Lys Thr Trp Glu Leu Thr Ala 145150 155 160 Ser Ala Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly His SerAsp 165 170 175 Thr Thr Val Ala Ile Ser Thr Ser Thr Val Leu Leu Cys GlyLeu Ser 180 185 190 Ala Val Ser Leu Leu Ala Cys Tyr Leu Lys Ser Arg AlaSer Val Cys 195 200 205 Ser Cys His Pro Arg Ser Ala Gly His Thr Cys SerVal Gly Ser Val 210 215 220 Cys 225 267 amino acids amino acid singlelinear Protein YES NO 15 Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr LeuGly Leu Pro Ala 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro AlaThr Arg Gly Ile Thr 20 25 30 Cys Pro Pro Pro Met Ser Val Glu His Ala AspIle Trp Val Lys Ser 35 40 45 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile CysAsn Ser Gly Phe Lys 50 55 60 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu CysVal Leu Asn Lys Ala 65 70 75 80 Thr Asn Val Ala His Trp Thr Thr Pro SerLeu Lys Cys Ile Arg Asp 85 90 95 Pro Ala Leu Val His Gln Arg Pro Ala ProPro Ser Thr Val Thr Thr 100 105 110 Ala Gly Val Thr Pro Gln Pro Glu SerLeu Ser Pro Ser Gly Lys Glu 115 120 125 Pro Ala Ala Ser Ser Pro Ser SerAsn Asn Thr Ala Ala Thr Thr Ala 130 135 140 Ala Ile Val Pro Gly Ser GlnLeu Met Pro Ser Lys Ser Pro Ser Thr 145 150 155 160 Gly Thr Thr Glu IleSer Ser His Glu Ser Ser His Gly Thr Pro Ser 165 170 175 Gln Thr Thr AlaLys Thr Trp Glu Leu Thr Ala Ser Val Ser His Gln 180 185 190 Pro Thr GlyVal Phe Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile 195 200 205 Ser ThrSer Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu 210 215 220 AlaCys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu 225 230 235240 Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg 245250 255 Asp Glu Asp Leu Glu Asn Cys Ser His His Leu 260 265

We claim:
 1. An isolated DNA encoding an IL-15 receptor (IL-15R),wherein the DNA is selected from the group consisting of: (a) a DNAhaving a nucleotide sequence as set forth in SEQ ID NO:1, nucleotides 91through 789; (b) a DNA having a nucleotide sequence as set forth in SEQID NO:6, nucleotides 34 through 753; (c) DNAs that hybridize to the DNAsequences of (a) or (b) or their complementary strands under conditionsof high stringency, and which encode polypeptides capable of bindingIL-15; and (d) DNAs that, due to degeneracy of the genetic code, encodepolypeptides encoded by any of the foregoing DNAs.
 2. An isolated DNAaccording to claim 1 that encodes a human IL-15R.
 3. An isolated DNAaccording to claim 1 that encodes a soluble IL-15R.
 4. An isolated DNAaccording to claim 3 wherein the DNA is selected from the groupconsisting of: (a) a DNA having a nucleotide sequence as set forth inSEQ ID NO:1, nucleotides 91 through 612; (b) a DNA having a nucleotidesequence as set forth in SEQ ID NO:6, nucleotides 34 through 567; (c) aDNA that encodes a fragment of a peptide encoded by the sequences of (a)or (b), which fragment is capable of binding IL-15; (d) DNAs thathybridize to the DNA sequences of (a), (b) or (c) or their complementarystrands under conditions of high stringency, and which encode apolypeptide capable of binding IL-15; and (e) DNAs that, due todegeneracy of the genetic code, encode a polypeptide encoded by any ofthe foregoing DNAs.
 5. An isolated DNA sequence according to claim 3that encodes a human IL-15R.
 6. An isolated DNA according to claim 1,encoding a modified IL-15R polypeptide having one or more changes in aprimary amino acid sequence, which changes are selected from the groupconsisting of: inactivated N-linked glycosylation sites; modified KEX2protease cleavage sites; deleted cysteine residues; and conservativeamino acid substitutions, wherein the modified polypeptide binds IL-15.7. A recombinant expression vector comprising a DNA according toclaim
 1. 8. A recombinant expression vector comprising a DNA accordingto claim
 4. 9. A recombinant expression vector comprising a DNAaccording to claim
 5. 10. A process for preparing an IL-15 receptor(IL-15R), comprising culturing a host cell transformed or transfectedwith a recombinant expression vector according to claim 7 underconditions promoting expression, and recovering a polypeptide from theculture, wherein the polypeptide is capable of binding IL-15.
 11. Aprocess for preparing an IL-15 receptor (IL-15R), comprising culturing ahost cell transformed or transfected with a recombinant expressionvector according to claim 8 under conditions promoting expression, andrecovering a polypeptide from the culture, wherein the polypeptide iscapable of binding IL-15.
 12. A process for preparing an IL-15 receptor(IL-15R), comprising culturing a host cell transformed or transfectedwith a recombinant expression vector according to claim 9 underconditions promoting expression, and recovering a polypeptide from theculture, wherein the polypeptide is capable of binding IL-15.
 13. Anisolated IL-15 receptor (IL-15R) encoded by a DNA sequence according toclaim
 1. 14. An IL-15 receptor (IL-15R) according to claim 13 which is asoluble IL-15R.
 15. An IL-15 receptor (IL-15R) according to claim 13,comprising a polypeptide selected from the group consisting of: (a) apolypeptide having an amino acid sequence as set forth in SEQ ID NO:2,having an amino terminus selected from the group consisting of aminoacid 31, amino acid 34 and amino acid 35 of SEQ ID NO:2, and a carboxyterminus selected from the group consisting of amino acid 204 and aminoacid 205 of SEQ ID NO:2; (b) a polypeptide having an amino acid sequenceas set forth in SEQ ID NO:6, having an amino terminus selected from thegroup consisting of amino acid 12, amino acid 15 and amino acid 16 ofSEQ ID NO:6, and a carboxy terminus selected from the group consistingof an amino acid between amino acid 78 and amino acid 189 of SEQ IDNO:6; and (c) a fragment of a polypeptide having an amino acid sequenceof the polypeptide of (a) or (b), which fragment is capable of bindingIL-15.
 17. Antibodies immunoreactive with mammalian IL-15 receptors. 18.An antibody according to claim 16 which is a monoclonal antibody.
 19. Anantibody according to claim 17 which is a blocking monoclonal antibody.